Anti-her2 antibody-drug conjugate

ABSTRACT

As an antitumor drug which is excellent in terms of antitumor effect and safety and has an excellent therapeutic effect, there is provided an antibody-drug conjugate in which an antitumor compound represented by the following formula is conjugated to an anti-HER2 antibody via a linker having a structure represented by the following formula: -L 1 -L 2 -L P -NH—(CH 2 )n 1 -L a -(CH 2 )n 2 —C(═O)— wherein the anti-HER2 antibody is connected to the terminal L 1 , and the antitumor compound is connected to the carbonyl group of the —(CH 2 )n 2 —C(═O)— moiety with the nitrogen atom of the amino group at position 1 as the connecting position.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application filed under 35 U.S.C.§111(a) claiming the benefit under 35 U.S.C. §§120 and 365(c) ofInternational Application No. PCT/JP2015/000355 filed on Jan. 28, 2015,which is based upon and claims the benefit of priority of JapanesePatent Application No. 2014-017777, filed on Jan. 31, 2014, JapanesePatent Application No. 2014-168944, filed on Aug. 22, 2014, and JapanesePatent Application No. 2014-227886, filed on Nov. 10, 2014, the contentsof which are hereby incorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to an antibody-drug conjugate having anantitumor drug conjugated to an anti-HER2 antibody via a linkerstructure moiety, the conjugate being useful as an antitumor drug.

BACKGROUND ART

An antibody-drug conjugate (ADC) having a drug with cytotoxicityconjugated to an antibody, whose antigen is expressed on the surface ofcancer cells and which also binds to an antigen capable of cellularinternalization, and therefore can deliver the drug selectively tocancer cells, is thus expected to cause accumulation of the drug withincancer cells and to kill the cancer cells (see, Non-patent Literatures 1to 3). As an ADC, Mylotarg (registered trademark; Gemtuzumab ozogamicin)in which calicheamicin is conjugated to an anti-CD33 antibody isapproved as a therapeutic agent for acute myeloid leukemia. Further,Adcetris (registered trademark; Brentuximab vedotin), in whichauristatin E is conjugated to an anti-CD30 antibody, has recently beenapproved as a therapeutic agent for Hodgkin's lymphoma and anaplasticlarge cell lymphoma (see, Non-patent Literature 4). The drugs containedin ADCs which have been approved until now target DNA or tubulin.

With regard to an antitumor agent, camptothecin derivatives,low-molecular-weight compounds that inhibit topoisomerase I to exhibitan antitumor effect, are known. Among these, an antitumor compoundrepresented by the formula below

(exatecan, chemical name:(1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10,13(9H,15H)-dione)is a water soluble derivative of camptothecin (Patent Literatures 1 and2). Unlike irinotecan currently used in clinical settings, this compounddoes not require activation by an enzyme for exhibiting its antitumoreffect. Further, its inhibitory activity on topoisomerase I was observedto be higher than SN-38 which is the main pharmaceutically activesubstance of irinotecan and topotecan also used in clinical settings,and higher in vitro cytocidal activity was confirmed against variouscancer cells. In particular, it was confirmed to have the effect againstcancer cells that have resistance to SN-38 or the like due to expressionof P-glycoprotein. Further, in a human tumor subcutaneously transplantedmouse model, it was confirmed to have a potent antitumor effect, andthus has undergone clinical studies, but has not been placed on themarket yet (see, Non-patent Literatures 5 to 10). It remains unclearwhether or not exatecan acts effectively as an ADC.

DE-310 is a complex in which exatecan is conjugated to a biodegradablecarboxymethyldextran polyalcohol polymer via a GGFG peptide spacer(Patent Literature 3). By converting exatecan into the form of a polymerprodrug, a high blood retention property can be maintained and also ahigh targeting property to tumor areas is passively increased byutilizing the increased permeability of newly formed blood vesselswithin tumors and retention property in tumor tissues. With DE-310,through cleavage of the peptide spacer by enzyme, exatecan and exatecanwith glycine connected to an amino group are continuously released asmain active substance, and as a result, the pharmacokinetics areimproved. DE-310 was found to have higher effectiveness than exatecanadministered alone even though the total dosage of exatecan contained inD310 is lower than in the case of administration of exatecan aloneaccording to various tumor evaluation models in non-clinical studies. Aclinical study was conducted for DE-310, and effective cases were alsoconfirmed, including a report suggesting that the main active substanceaccumulates in tumors more than in normal tissues. However, there isalso a report indicating that accumulation of DE-310 and the main activesubstance in tumors is not much different from accumulation in normaltissues in humans, and thus no passive targeting is observed in humans(see, Non-patent Literatures 11 to 14). As a result, DE-310 was not alsocommercialized, and it remains unclear whether or not exatecaneffectively acts as a drug directed to such targeting.

As a compound relating to DE-310, a complex in which a structure moietyrepresented by —NH—(CH₂)₄—C(═O)— is inserted between the -GGFG- spacerand exatecan to form -GGFG-NH—(CH₂)₄—C(═O)— used as a spacer structureis also known (Patent Literature 4). However, the antitumor effect ofsaid complex is not known at all.

HER2 is one of the products of a typical growth factor receptor typeoncogene identified as human epidermal cell growth factor receptor2-related oncogene, and is a transmembrane receptor protein having amolecular weight of 185 kDa and having a tyrosine kinase domain(Non-patent Literature 15). The DNA sequence and amino acid sequence ofHER2 are disclosed on a public database, and can be referred to, forexample, under Accession No. M11730 (GenBank), NP_004439.2 (NCBI), orthe like.

HER2 (neu, ErbB-2) is one of the members of the EGFR (epidermal growthfactor receptor) family and is activated by autophosphorylation atintracellular tyrosine residues by its homodimer formation orheterodimer formation with another EGFR receptor HER1 (EGFR, ErbB-1),HER3 (ErbB-3), or HER4 (ErbB-4) (Non-patent Literatures 16 to 18),thereby playing an important role in cell growth, differentiation, andsurvival in normal cells and cancer cells (Non-patent Literatures 19 and20). HER2 is overexpressed in various cancer types such as breastcancer, gastric cancer, and ovarian cancer (Non-patent Literatures 21 to26) and has been reported to be a negative prognosis factor for breastcancer (Non-patent Literatures 27 and 28).

Trastuzumab is a humanized antibody of a mouse anti-HER2 antibody 4D5(Non-patent Literature 29 and Patent Literature 5), named as recombinanthumanized anti-HER2 monoclonal antibody (huMAb4D5-8, rhuMAb HER2,Herceptin(R)) (Patent Literature 6). Trastuzumab specifically binds tothe extracellular domain IV of HER2 and induces antibody-dependentcellular cytotoxicity (ADCC) or exerts an anticancer effect via theinhibition of signal transduction from HER2 (Non-patent Literatures 30and 31). Trastuzumab is highly effective for tumors overexpressing HER2(Non-patent Literature 32) and as such, was launched in 1999 in the USAand in 2001 in Japan as a therapeutic agent for patients with metastaticbreast cancer overexpressing HER2.

Although the therapeutic effect of trastuzumab on breast cancer has beenadequately proven (Non-patent Literature 33), allegedly about 15% ofpatients with breast cancer overexpressing HER2 who have received a widerange of conventional anticancer therapies are responders totrastuzumab. About 85% of patients of this population have no or merelyweak response to trastuzumab treatment.

Thus, the need for a therapeutic agent targeting HER2 expression-relateddiseases has been recognized for patients affected by tumorsoverexpressing HER2 with no or weak response to trastuzumab orHER2-related disorders. T-DM1 (trastuzumab emtansine, Kadcyla (R);Non-patent Literature 34) having an antitumor drug conjugated totrastuzumab via a linker structure, and pertuzumab (Perjeta(R);Non-patent Literature 35 and Patent Literature 7) designed to target theextracellular domain II of HER2 and inhibit heterodimer formation havebeen developed. However, their responsiveness, activity strength, andaccepted indications are still insufficient, and there are unsatisfiedneeds for targeting HER2.

CITATION LIST Patent Literatures

-   [Patent Literature 1] Japanese Patent Laid-Open No. 5-59061-   [Patent Literature 2] Japanese Patent Laid-Open No. 8-337584-   [Patent Literature 3] International Publication No. WO 1997/46260-   [Patent Literature 4] International Publication No. WO 2000/25825-   [Patent Literature 5] U.S. Pat. No. 5,677,171-   [Patent Literature 6] U.S. Pat. No. 5,821,337-   [Patent Literature 7] International Publication No. WO 01/00244

Non-Patent Literatures

-   [Non-patent Literature 1] Ducry, L., et al., Bioconjugate    Chem. (2010) 21, 5-13.-   [Non-patent Literature 2] Alley, S. C., et al., Current Opinion in    Chemical Biology (2010) 14, 529-537.-   [Non-patent Literature 3] Damle N. K. Expert Opin. Biol.    Ther. (2004) 4, 1445-1452.-   [Non-patent Literature 4] Senter P. D., et al., Nature    Biotechnology (2012) 30, 631-637.-   [Non-patent Literature 5] Kumazawa, E., Tohgo, A., Exp. Opin.    Invest. Drugs (1998) 7, 625-632.-   [Non-patent Literature 6] Mitsui, I., et al., Jpn J. Cancer    Res. (1995) 86, 776-782.-   [Non-patent Literature 7] Takiguchi, S., et al., Jpn J. Cancer    Res. (1997) 88, 760-769.-   [Non-patent Literature 8] Joto, N. et al. Int J Cancer (1997) 72,    680-686.-   [Non-patent Literature 9] Kumazawa, E. et al., Cancer Chemother.    Pharmacol. (1998) 42, 210-220.-   [Non-patent Literature 10] De Jager, R., et al., Ann NY Acad    Sci (2000) 922, 260-273.-   [Non-patent Literature 11] Inoue, K. et al., Polymer Drugs in the    Clinical Stage, Edited by Maeda et al. (2003) 145-153.-   [Non-patent Literature 12] Kumazawa, E. et al., Cancer Sci (2004)    95, 168-175.-   [Non-patent Literature 13] Soepenberg, O. et al., Clinical Cancer    Research, (2005) 11, 703-711.-   [Non-patent Literature 14] Wente M. N. et al., Investigational New    Drugs (2005) 23, 339-347.-   [Non-patent Literature 15] Coussens L, et al., Science. 1985;    230(4730):1132-1139.-   [Non-patent Literature 16] Graus-Porta G, et al., EMBO J. 1997; 16;    1647-1655.-   [Non-patent Literature 17] Karunagaran D, et al., EMBO J. 1996;    15:254-264.-   [Non-patent Literature 18] Sliwkowski M X, et al., J. Biol. Chem.    1994; 269:14661-14665.-   [Non-patent Literature 19] Di Fore P P, et al., Science. 1987;    237:178-182.-   [Non-patent Literature 20] Hudziak R M, et al., Proc Natl Acad Sci    USA. 1987; 84:7159-7163.-   [Non-patent Literature 21] Hardwick R, et al., Eur. J Surg Oncol.    1997 (23):30-35.-   [Non-patent Literature 22] Korkaya H, et al., Oncogene. 2008;    27(47):6120-6130.-   [Non-patent Literature 23] Yano T, et al., Oncol Rep. 2006;    15(1):65-71.-   [Non-patent Literature 24] Slamon D J, et al., Science. 1987;    235:177-182.-   [Non-patent Literature 25] Gravalos C, et al., Ann Oncol 19:    1523-1529, 2008.-   [Non-patent Literature 26] Fukushige S et al., Mol Cell Biol 6:    955-958, 1986.-   [Non-patent Literature 27] Slamon D J, et al. Science. 1989;    244:707-712.-   [Non-patent Literature 28] Kaptain S et al., Diagn Mol Pathol    10:139-152, 2001.-   [Non-patent Literature 29] Fendly. et al., Cancer Research    1990(50):1550-1558.-   [Non-patent Literature 30] Sliwkowski M X, et al., Semin Oncol.    1999; 26(4, Suppl 12):60-70.-   [Non-patent Literature 31] Hudis C A, et al., N Engl J Med. 357:    39-51, 2007.-   [Non-patent Literature 32] Vogel C L, et al., J Clin Oncol. 2002;    20(3):719-726.-   [Non-patent Literature 33] Baselga et al., J. Clin. Oncol.    14:737-744 (1996).-   [Non-patent Literature 34] Burris III et al., J Clin Oncol 2011;    29:398-405.-   [Non-patent Literature 35] Adams C W, et al., Cancer Immunol    Immunother. 2006; 6:717-727.

SUMMARY OF INVENTION Technical Problem

With regard to the treatment of tumors by antibodies, an insufficientantitumor effect may be observed even when the antibody recognizes anantigen to bind to tumor cells, and there are cases in which a moreeffective antitumor antibody is needed. Further, many antitumorlow-molecular-weight compounds have problems in safety like side effectsand toxicity even if the compounds have an excellent antitumor effect.It has remained an objective to achieve a superior therapeutic effect byfurther enhancing safety. Thus, an object of the present invention is toprovide an antitumor drug having an excellent therapeutic effect, whichis excellent in terms of antitumor effect and safety.

Solution to Problem

The inventors considered that an anti-HER2 antibody is an antibody whichis capable of targeting tumor cells, that is, having a property ofrecognizing tumor cells, a property of binding to tumor cells, aproperty of internalizing within tumor cells, a cytotoxic activityagainst tumor cells, a cytocidal activity against tumor cells, or thelike; thus, when the antitumor compound exatecan is converted into anantibody-drug conjugate, via a linker structure moiety, by conjugationto this antibody, the antitumor compound can be more surely delivered totumor cells to specifically exhibit the antitumor effect of the compoundin tumor cells, and thus the antitumor effect can be surely exhibitedand also an enhanced cytocidal effect of the anti-HER2 antibody can beexpected, and the dose of the antitumor compound can be reduced comparedto the case of administering the compound alone, and thus influences ofthe antitumor compound on normal cells can be alleviated so that ahigher safety can be achieved.

In this connection, the inventors created a linker with a specificstructure and succeeded in obtaining an antibody-drug conjugate in whichthe anti-HER2 antibody and exatecan are conjugated to each other via thelinker, and confirmed an excellent antitumor effect exhibited by theconjugate to thereby complete the present invention.

Specifically, the present invention relates to the following.

[1] An antibody-drug conjugate wherein an antitumor compound representedby the following formula:

is conjugated to an anti-HER2 antibody via a linker having a structurerepresented by the following formula:

-L¹-L²-L^(P)-NH—(CH₂)n ¹-L^(a)-(CH₂)n ²-C(═O)—

via a thioether bond which is formed at a disulfide bond moiety presentin the hinge part of the anti-HER2 antibody.

Here, the anti-HER2 antibody is connected to the terminal L¹,

the antitumor compound is connected to the carbonyl group of the—(CH₂)n²-C(═O)— moiety with the nitrogen atom of the amino group atposition 1 as the connecting position,whereinn¹ represents an integer of 0 to 6,n² represents an integer of 0 to 5,L represents -(Succinimid-3-yl-N)—(CH₂)n³-C(═O)—,

wherein n³ represents an integer of 2 to 8,

-   L² represents —NH—(CH₂CH₂—O)n⁴-CH₂CH₂—C(═O)— or a single bond,

wherein n⁴ represents an integer of 1 to 6,

L^(P) represents a peptide residue consisting of 2 to 7 amino acids,L^(a) represents —O— or a single bond, and -(Succinimid-3-yl-N)— has astructure represented by the following formula:

which is connected to the anti-HER2 antibody at position 3 thereof andis connected to the methylene group in the linker structure containingthis structure on the nitrogen atom at position 1.

The present invention further relates to each of the following.

[2] The antibody-drug conjugate according to [1], wherein the peptideresidue L^(P) is a peptide residue comprising an amino acid selectedfrom phenylalanine, glycine, valine, lysine, citrulline, serine,glutamic acid, and aspartic acid.[3] The antibody-drug conjugate according to [1] or [2], wherein L^(P)is a peptide residue selected from the following group:

-GGF-, -DGGF-, -(D-)D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-,-GGFGG-, -DDGGFG-, -KDGGFG-, and -GGFGGGF-;

wherein “(D-)D” represents D-aspartic acid.[4] The antibody-drug conjugate according to [1] or [2], wherein L^(P)is a peptide residue consisting of 4 amino acids.[5] The antibody-drug conjugate according to any one of [1] to [4],wherein L^(P) is the tetrapeptide residue -GGFG-.[6] The antibody-drug conjugate according to any one of [1] to [5],wherein n³ is an integer of 2 to 5, and L² is a single bond.[7] The antibody-drug conjugate according to any one of [1] to [5],wherein n³ is an integer of 2 to 5, L² is —NH—(CH₂CH₂—O)n⁴-CH₂CH₂—C(═O)—, and n⁴ is 2 or 4.[8] The antibody-drug conjugate according to any one of [1] to [7],wherein —NH—(CH₂)n¹-L^(a)-(CH₂)n²-C(═O)— is a partial structure having achain length of 4 to 7 atoms.[9] The antibody-drug conjugate according to any one of [1] to [7],wherein —NH—(CH₂)n¹-L^(a)-(CH₂)n²-C(═O)— is a partial structure having achain length of 5 or 6 atoms.[10] The antibody-drug conjugate according to any one of [1] to [9],wherein —NH—(CH₂)n¹-L^(a)-(CH₂)n²-C(═O)— is—NH—CH₂CH₂—C(═O)—,—NH—CH₂CH₂CH₂—C(═O)—,—NH—CH₂CH₂CH₂CH₂—C(═O)—,—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—,—NH—CH₂—O—CH₂—C(═O)—,—NH—CH₂CH₂—O—CH₂—C(═O)—, or—NH—CH₂CH₂—O—C(═O)—.[11] The antibody-drug conjugate according to any one of [1] to [9],wherein —NH—(CH₂)n¹-L^(a)-(CH₂)n²-C(═O)— is—NH—CH₂CH₂CH₂—C(═O)—,—NH—CH₂—O—CH₂—C(═O)—, or—NH—CH₂CH₂—O—CH₂—C(═O)—.[12] The antibody-drug conjugate according to any one of [1] to [9],wherein the drug-linker structure moiety having the drug connected to-L¹-L²-L^(P)-NH— (CH₂)n¹-L^(a)-(CH₂)n²-C(═O)— is one drug-linkerstructure selected from the following group:-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂HH₂—O—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),wherein -(Succinimid-3-yl-N)— has a structure represented by thefollowing formula:

which is connected to the anti-HER2 antibody at position 3 thereof andis connected to the methylene group in the linker structure containingthis structure on the nitrogen atom at position 1,—(NH-DX) represents a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is theconnecting position, and -GGFG- represents the tetrapeptide residue-Gly-Gly-Phe-Gly-.[13] The antibody-drug conjugate according to any one of [1] to [9],wherein the drug-linker structure moiety having the drug connected to-L¹-L²-L-NH—(CH₂)n¹-L^(a)-(CH₂)n²-C(═O)— is one drug-linker structureselected from the following group:-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂CH—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)

Here, -(Succinimid-3-yl-N)—, —(NH-DX), and -GGFG- are as defined above.

[14] An antibody-drug conjugate wherein an antitumor compoundrepresented by the following formula:

is conjugated to an anti-HER2 antibody via a linker having a structurerepresented by the following formula:

-L¹-L²-L^(P)-NH—(CH₂)n ¹-L^(a)-(CH₂)n ²-C(═O)—

via a thioether bond which is formed at a disulfide bond moiety presentin the hinge part of the anti-HER2 antibody,whereinthe anti-HER2 antibody is connected to the terminal L¹, the antitumorcompound is connected to the carbonyl group of the —(CH₂)n²-C(═O)—moiety,whereinn¹ represents an integer of 0 to 6,n² represents an integer of 0 to 5,L represents -(Succinimid-3-yl-N)—(CH₂)n³-C(═O)—,

wherein n³ represents an integer of 2 to 8,

L² represents —NH—(CH₂CH₂—O) n⁴-CH₂CH₂—C(═O)— or a single bond,

wherein n⁴ represents an integer of 1 to 6,

L^(P) represents the tetrapeptide residue -GGFG-,L^(a) represents —O— or a single bond, and-(Succinimid-3-yl-N)— has a structure represented by the followingformula:

which is connected to the anti-HER2 antibody at position 3 thereof andis connected to the methylene group in the linker structure containingthis structure on the nitrogen atom at position 1.[15] The antibody-drug conjugate according to [14], whereinn¹ is 3, n² is 0, n³ is 2, L² is —NH—(CH₂CH₂—O)n⁴—CH₂CH₂—C(═O)—, n⁴ is2, and L^(a) is a single bond,n¹ is 1, n² is 1, n³ is 5, L² is a single bond, and L^(a) is —O—, orn¹ is 2, n² is 1, n³ is 5, L² is a single bond, and L^(a) is —O—.[16] The antibody-drug conjugate according to [14] or [15], wherein n³is 2 or 5, and L² is a single bond.[17] The antibody-drug conjugate according to [14] or [15], wherein n³is 2 or 5, L² is —NH—(CH₂CH₂—O) n⁴—CH₂CH₂—C(═O)—, and n⁴ is 2 or 4.[18] The antibody-drug conjugate according to any one of [14] to [17],wherein —NH—(CH₂) n-L^(a)-(CH₂)n²—C(═O)— is—NH—CH₂CH₂CH₂—C(═O)—,—NH—CH₂—O—CH₂—C(═O)—, or—NH—CH₂CH₂—O—CH₂—C(═O)—.[19] The antibody-drug conjugate according to any one of [14] to [18],wherein the drug-linker structure moiety having the drug connected to-L-L²-L^(P)-NH—(CH₂)n¹-L^(a)-(CH₂)n²—C(═O)— is one drug-linker structureselected from the group consisting of the following:-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂HH₂—O—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),

wherein, -(Succinimid-3-yl-N)— has a structure represented by thefollowing formula:

which is connected to the anti-HER2 antibody at position 3 thereof andis connected to the methylene group in the linker structure containingthis structure on the nitrogen atom at position 1,—(NH-DX) represents a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is theconnecting position, and-GGFG- represents the tetrapeptide residue -Gly-Gly-Phe-Gly-.[20] The antibody-drug conjugate according to any one of [14] to [18],wherein the drug-linker structure moiety having the drug connected to-L¹-L²-L^(P)-NH—(CH₂)n¹-L^(a)-(CH₂)n²—C(═O)— is one drug-linkerstructure selected from the following group:-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),and-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX).

Here, -(Succinimid-3-yl-N)—, —(NH-DX), and -GGFG- are as defined above.

[21] The antibody-drug conjugate according to any one of [1] to [20],wherein the average number of units of the selected one drug-linkerstructure conjugated per antibody molecule is in the range of from 1 to10.[22] The antibody-drug conjugate according to any one of [1] to [20],wherein the average number of units of the selected one drug-linkerstructure conjugated per antibody molecule is in the range of from 2 to8.[23] The antibody-drug conjugate according to any one of [1] to [20],wherein the average number of units of the selected one drug-linkerstructure conjugated per antibody molecule is in the range of from 3 to8.[24] A drug containing the antibody-drug conjugate according to any oneof [1] to [23], a salt thereof or a hydrate thereof.[25] An antitumor drug and/or anticancer drug containing theantibody-drug conjugate according to any one of [1] to [23], a saltthereof or a hydrate thereof.[26] The antitumor drug and/or anticancer drug according to [25], whichis for use against lung cancer, urothelial cancer, colorectal cancer,prostate cancer, ovarian cancer, pancreatic cancer, breast cancer,bladder cancer, gastric cancer, gastrointestinal stromal tumor, uterinecervix cancer, esophageal cancer, squamous cell carcinoma, peritonealcancer, liver cancer, hepatocellular cancer, colon cancer, rectalcancer, colorectal cancer, endometrial cancer, uterine cancer, salivarygland cancer, kidney cancer, vulval cancer, thyroid cancer, peniscancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.[27] A pharmaceutical composition containing the antibody-drug conjugateaccording to any one of [1] to[23], a salt thereof or a hydrate thereof as an active component, and apharmaceutically acceptable formulation component.[28] The pharmaceutical composition according to [27], which is for useagainst lung cancer, urothelial cancer, colorectal cancer, prostatecancer, ovarian cancer, pancreatic cancer, breast cancer, bladdercancer, gastric cancer, gastrointestinal stromal tumor, uterine cervixcancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer,liver cancer, hepatocellular cancer, colon cancer, rectal cancer,colorectal cancer, endometrial cancer, uterine cancer, salivary glandcancer, kidney cancer, vulval cancer, thyroid cancer, penis cancer,leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.[29] A method for treating tumor and/or cancer comprising administeringthe antibody-drug conjugate according to any one of [1] to [23], a saltthereof or a hydrate thereof.[30] A method for producing an antibody-drug conjugate comprisingreacting a compound represented by the following formula:

(maleimid-N-yl)-(CH₂)n ³—C(═O)-L²-L¹-NH—(CH₂)n ¹-L^(a)-(CH₂)n²—C(═O)—(NH-DX)

with an anti-HER2 antibody or a reactive derivative thereof andconjugating a drug-linker moiety to the antibody by a method for forminga thioether bond at a disulfide bond site present in the hinge part ofthe antibody.

In the formula, n³ represents an integer of 2 to 8, L² represents—NH—(CH₂CH₂—O)n⁴—CH₂CH₂—C(═O)— or a single bond,

wherein n⁴ represents an integer of 1 to 6,

L^(P) represents a peptide residue consisting of 2 to 7 amino acidsselected from phenylalanine, glycine, valine, lysine, citrulline,serine, glutamic acid, and aspartic acid,n¹ represents an integer of 0 to 6,n² represents an integer of 0 to 5,L^(a) represents —O— or a single bond,(maleimid-N-yl)- is a group represented by the following formula:

wherein the nitrogen atom is the connecting position, and—(NH-DX) is a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is theconnecting position.[31] The production method according to [30], wherein the method forconjugating a drug-linker moiety to an anti-HER2 antibody is a method ofreducing the antibody to convert the antibody to a reactive derivative.[32] The production method according to [30] or [31], wherein theaverage number of units of the selected one drug-linker structureconjugated per antibody molecule is in the range of from 1 to 10.[33] The production method according to [30] or [31], wherein theaverage number of units of the selected one drug-linker structureconjugated per antibody molecule is in the range of from 2 to 8.[34] The production method according to [30] or [31], wherein theaverage number of units of the selected one drug-linker structureconjugated per antibody molecule is in the range of from 3 to 8.[35] An antibody-drug conjugate obtained by the production methodaccording to any of [30] to [34].[36] An antibody-drug conjugate obtained by forming a thioether bond ata sulfide bond site in the hinge part of the antibody, wherein theanti-HER2 antibody is treated in a reducing condition and thereafterreacted with a compound selected from the group shown below:(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),and(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX).

In the above, (maleimid-N-yl)- is a group represented by the followingformula:

wherein the nitrogen atom is the connecting position, and—(NH-DX) is a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is theconnecting position, and-GGFG- represents the tetrapeptide residue -Gly-Gly-Phe-Gly-.[37] An antibody-drug conjugate obtained by forming a thioether bond ata sulfide bond site present in the hinge part of the antibody, whereinthe anti-HER2 antibody is treated in a reducing condition and thereafterreacted with a compound selected from the group shown below:(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),and(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX).

Here, (maleimid-N-yl)-, —(NH-DX), and -GGFG- are as defined above.

[38] The antibody-drug conjugate according to [36] or [37], wherein theaverage number of units of the selected one drug-linker structureconjugated per antibody molecule is in the range of from 1 to 10.[39] The antibody-drug conjugate according to [36] or [37], wherein theaverage number of units of the selected one drug-linker structureconjugated per antibody molecule is in the range of from 2 to 8.[40] The antibody-drug conjugate according to [36] or [37], wherein theaverage number of units of the selected one drug-linker structureconjugated per antibody molecule is in the range of from 3 to 8.

Advantageous Effects of Invention

With an anti-HER2 antibody-drug conjugate having the antitumor compoundexatecan conjugated via a linker with a specific structure, an excellentantitumor effect and safety can be achieved.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an amino acid sequence of a heavy chain of a humanizedanti-HER2 monoclonal antibody (SEQ ID NO: 1).

FIG. 2 shows an amino acid sequence of a light chain of a humanizedanti-HER2 monoclonal antibody (SEQ ID NO: 2).

FIG. 3 is a diagram showing the antitumor effect of an antibody-drugconjugate (27) or trastuzumab on a nude mouse with subcutaneouslytransplanted human breast cancer line KPL-4 cells. In the drawing, theabscissa depicts days after tumor inoculation, and the ordinate depictstumor volume.

FIG. 4 is a diagram showing the antitumor effect of an antibody-drugconjugate (8), (28) or trastuzumab emtansine on a nude mouse withsubcutaneously transplanted human gastric cancer line NCI-N87 cells. Inthe drawing, the abscissa depicts days after tumor inoculation, and theordinate depicts tumor volume.

FIG. 5 is a diagram showing the antitumor effect of an antibody-drugconjugate (8), (29), (30), trastuzumab, or trastuzumab emtansine on anude mouse with subcutaneously transplanted human breast cancer lineJIMT-1 cells. In the drawing, the abscissa depicts days after tumorinoculation, and the ordinate depicts tumor volume.

FIG. 6 is a diagram showing the antitumor effect of an antibody-drugconjugate (31), trastuzumab, or trastuzumab emtansine on a nude mousewith subcutaneously transplanted human pancreatic cancer line Capan-1cells. In the drawing, the abscissa depicts days after tumorinoculation, and the ordinate depicts tumor volume.

FIG. 7 is a diagram showing the antitumor effect of an antibody-drugconjugate (50) on a nude mouse with subcutaneously transplanted humangastric cancer line NCI-N87 cells. In the drawing, the abscissa depictsdays after tumor inoculation, and the ordinate depicts tumor volume.

FIG. 8 is a diagram showing the antitumor effect of an antibody-drugconjugate (50), trastuzumab, or trastuzumab emtansine on a nude mousewith subcutaneously transplanted human breast cancer ST225 cells. In thedrawing, the abscissa depicts days after tumor inoculation, and theordinate depicts tumor volume.

FIG. 9 is a diagram showing the antitumor effect of an antibody-drugconjugate (50), trastuzumab, or trastuzumab emtansine on a nude mousewith subcutaneously transplanted human breast cancer ST910 cells. In thedrawing, the abscissa depicts days after tumor inoculation, and theordinate depicts tumor volume.

FIG. 10 is a diagram showing the antitumor effect of an antibody-drugconjugate (50), trastuzumab, or trastuzumab emtansine on a nude mousewith subcutaneously transplanted human colorectal cancer line CTG-0401cells. In the drawing, the abscissa depicts days after tumorinoculation, and the ordinate depicts tumor volume.

FIG. 11 is a diagram showing the antitumor effect of an antibody-drugconjugate (50), trastuzumab, or trastuzumab emtansine on a nude mousewith subcutaneously transplanted human non-small cell lung cancerCTG-0860 cells. In the drawing, the abscissa depicts days after tumorinoculation, and the ordinate depicts tumor volume.

FIG. 12 is a diagram showing the antitumor effect of an antibody-drugconjugate (50), trastuzumab, or trastuzumab emtansine on a nude mousewith subcutaneously transplanted human bile duct cancer line CTG-0927cells. In the drawing, the abscissa depicts days after tumorinoculation, and the ordinate depicts tumor volume.

FIG. 13 is a diagram showing the antitumor effect of an antibody-drugconjugate (50), trastuzumab, or trastuzumab emtansine on a nude mousewith subcutaneously transplanted human esophageal cancer line CTG-0137cells. In the drawing, the abscissa depicts days after tumorinoculation, and the ordinate depicts tumor volume.

FIG. 14 is a diagram showing the antitumor effect of an antibody-drugconjugate (50), trastuzumab, or trastuzumab emtansine on a nude mousewith subcutaneously transplanted human ovarian cancer line SK-OV-3cells. In the drawing, the abscissa depicts days after tumorinoculation, and the ordinate depicts tumor volume.

DESCRIPTION OF EMBODIMENTS

Hereinafter, preferred modes for carrying out the present invention aredescribed with reference to the drawings. The embodiments describedbelow are given merely for illustrating one example of a typicalembodiment of the present invention and are not intended to limit thescope of the present invention.

The anti-HER2 antibody-drug conjugate of the present invention is anantitumor drug in which an anti-HER2 antibody is conjugated to anantitumor compound via a linker structure moiety and is explained indetail hereinbelow.

[Antibody]

The anti-HER2 antibody used in the anti-HER2 antibody-drug conjugate ofthe present invention may be derived from any species, and preferredexamples of the species can include humans, rats, mice, and rabbits. Incase when the antibody is derived from other than human species, it ispreferably chimerized or humanized using a well known technique. Theantibody of the present invention may be a polyclonal antibody or amonoclonal antibody and is preferably a monoclonal antibody.

The anti-HER2 antibody is the antibody, which is capable of targetingtumor cells, that is, possesses a property of recognizing a tumor cell,a property of binding to a tumor cell, a property of internalizing in atumor cell, cytocidal activity against tumor cells, or the like, and canbe conjugated with a drug having antitumor activity via a linker to forman antibody-drug conjugate.

The binding activity of the antibody against tumor cells can beconfirmed using flow cytometry. The internalization of the antibody intotumor cells can be confirmed using (1) an assay of visualizing anantibody incorporated in cells under a fluorescence microscope using asecondary antibody (fluorescently labeled) binding to the therapeuticantibody (Cell Death and Differentiation (2008) 15, 751-761), (2) anassay of measuring a fluorescence intensity incorporated in cells usinga secondary antibody (fluorescently labeled) binding to the therapeuticantibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December2004), or (3) a Mab-ZAP assay using an immunotoxin binding to thetherapeutic antibody wherein the toxin is released upon incorporationinto cells to inhibit cell growth (Bio Techniques 28: 162-165, January2000). As the immunotoxin, a recombinant complex protein of a diphtheriatoxin catalytic domain and protein G may be used.

The antitumor activity of the antibody can be confirmed in vitro bydetermining inhibitory activity against cell growth. For example, acancer cell line overexpressing a target protein for the antibody iscultured, and the antibody is added at varying concentrations into theculture system to determine an inhibitory activity against focusformation, colony formation, and spheroid growth. The antitumor activitycan be confirmed in vivo, for example, by administering the antibody toa nude mouse with a transplanted tumor cell line highly expressing thetarget protein, and determining change in the cancer cell.

Since the compound conjugated in the antibody-drug conjugate exerts anantitumor effect, it is preferred but not essential that the antibodyitself should have an antitumor effect. For the purpose of specificallyand selectively exerting the cytotoxic activity of the antitumorcompound against tumor cells, it is important and also preferred thatthe antibody should have the property of internalizing to migrate intotumor cells.

The anti-HER2 antibody can be obtained by a procedure known in the art.For example, the antibody of the present invention can be obtained usinga method usually carried out in the art, which involves immunizinganimals with an antigenic polypeptide and collecting and purifyingantibodies produced in vivo. The origin of the antigen is not limited tohumans, and the animals may be immunized with an antigen derived from anon-human animal such as a mouse, a rat and the like. In this case, thecross-reactivity of antibodies binding to the obtained heterologousantigen with human antigens can be tested to screen for an antibodyapplicable to a human disease.

Alternatively, antibody-producing cells which produce antibodies againstthe antigen are fused with myeloma cells according to a method known inthe art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; andKennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y.(1980)) to establish hybridomas, from which monoclonal antibodies can inturn be obtained.

The antigen can be obtained by genetically engineering host cells toproduce a gene encoding the antigenic protein. Specifically, vectorsthat permit expression of the antigen gene are prepared and transferredto host cells so that the gene is expressed. The antigen thus expressedcan be purified. The antibody can also be obtained by a method ofimmunizing animals with the above-described genetically engineeredantigen-expressing cells or a cell line expressing the antigen.

The anti-HER2 antibodies that can be used in the present invention arenot particularly limited and are preferably, for example, those havingproperties as described below.

(1) An anti-HER2 antibody having the following properties:

(a) specifically binding to HER2, and

(b) having an activity of internalizing in HER2-expressing cells bybinding to HER2.

(2) The antibody according to (1) above, wherein the antibody binds tothe extracellular domain of HER2.(3) The antibody according to (1) or (2) above, wherein the antibody isa monoclonal antibody.(4) The antibody according to any of (1) to (3) above, wherein theantibody has an antibody-dependent cellular cytotoxicity (ADCC) activityand/or a complement-dependent cytotoxicity (CDC) activity(5) The antibody according to any of (1) to (4) above, wherein theantibody is a mouse monoclonal antibody, a chimeric monoclonal antibody,or a humanized monoclonal antibody.(6) The antibody according to any of (1) to (5) above, wherein theantibody is a humanized monoclonal antibody comprising a heavy chainconsisting of the amino acid sequence represented by SEQ ID NO: 1 and alight chain consisting of the amino acid sequence represented by SEQ IDNO: 2.(7) The antibody according to any of (1) to (6) above, wherein theantibody lacks a lysine residue at the carboxyl terminus of the heavychain.(8) The antibody according to (7) above, wherein the antibody comprisesa heavy chain consisting of an amino acid sequence consisting of aminoacid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting ofan amino acid sequence consisting of amino acid residues 1 to 214 of SEQID NO: 2.(9) An antibody obtained by a method for producing the antibodyaccording to any of (1) to (8) above, the method comprising the stepsof: culturing a host cell transformed with an expression vectorcontaining a polynucleotide encoding the antibody; and collecting theantibody of interest from the cultures obtained in the preceding step.

Hereinafter, the anti-HER2 antibody used in the invention is described.

The terms “cancer” and “tumor” as used herein are used with the samemeaning.

The term “gene” as used herein includes not only DNA, but also mRNAthereof, cDNA thereof and cRNA thereof.

The term “polynucleotide” as used herein is used with the same meaningas a nucleic acid and also includes DNA, RNA, probes, oligonucleotides,and primers.

The terms “polypeptide”, “protein” and “protein” as used herein are usedwithout distinction.

The term “cell” as used herein also includes cells in an animalindividual and cultured cells.

The term “HER2” as used herein is used with the same meaning as HER2protein.

Examples of the anti-HER2 antibody as used herein can include, but notparticularly limited to, pertuzumab (International Patent PublicationNo. WO 01/00245) and trastuzumab (U.S. Pat. No. 5,821,337). Trastuzumabis preferred. However, the anti-HER2 antibody of the present inventionis not limited thereto as long as it is an anti-HER2 antibodyspecifically binding to HER2, and more preferably having an activity ofinternalizing in HER2-expressing cells by binding to HER2.

The term “trastuzumab” as used herein is also called HERCEPTIN(R),huMAb4D5-8, or rhuMAb4D5-8 and is a humanized antibody comprising aheavy chain consisting of an amino acid sequence consisting of aminoacid residues 1 to 449 of SEQ ID NO: 1 (FIG. 1) and a light chainconsisting of an amino acid sequence consisting of amino acid residues 1to 214 of SEQ ID NO: 2 (FIG. 2).

The term “specifically binding” as used herein means binding that is notnonspecific adsorption. Examples of the criterion for determiningwhether the binding is specific or not can include dissociation constant(hereinafter referred to as “KD”). The KD value of the antibody for theHER2 protein is preferably 1×10⁻⁵ M or smaller, 5×10⁻⁶ M or smaller,2×10⁻⁶ M or smaller, or 1×10⁻⁶ M or smaller, more preferably 5×10⁻⁷ M orsmaller, 2×10⁻⁷ M or smaller, or 1×10⁻⁷ M or smaller, further preferably5×10⁻⁸ M or smaller, 2×10⁻⁸ M or smaller, or 1×10⁻⁸ M or smaller, andmost preferably 5×10⁻⁹ M or smaller, 2×10⁻⁹ M or smaller, or 1×10⁻⁹ M orsmaller. The binding between the HER2 protein and the antibody can bemeasured using a method known in the art, such as surface plasmonresonance, ELISA, or RIA.

The term “CDR” as used herein refers to a complementarity determiningregion (CDR). It is known that each heavy and light chain of an antibodymolecule has three complementarity determining regions (CDRs). The CDRis also called the hypervariable domain, and is present in a variableregion of each heavy and light chain of an antibody. It is a site whichhas unusually high variability in its primary structure, and there arethree separate CDRs in the primary structure of each heavy and lightpolypeptide chain. In this specification, as for the CDRs of anantibody, the CDRs of the heavy chain are represented by CDRH1, CDRH2,and CDRH3 from the amino-terminal side of the amino acid sequence of theheavy chain, and the CDRs of the light chain are represented by CDRL1,CDRL2, and CDRL3 from the amino-terminal side of the amino acid sequenceof the light chain. These sites are proximate to one another in thetertiary structure and determine the specificity for an antigen to whichthe antibody binds. The phrase “hybridization is performed understringent conditions” as used herein refers to a process in whichhybridization is performed under conditions under which identificationcan be achieved by performing hybridization at 68° C. in a commerciallyavailable hybridization solution ExpressHyb Hybridization Solution(manufactured by Clontech, Inc.) or by performing hybridization at 68°C. in the presence of 0.7 to 1.0 M NaCl using a filter having DNAimmobilized thereon, followed by performing washing at 68° C. using 0.1to 2×SSC solution (1×SSC solution is composed of 150 mM NaCl and 15 mMsodium citrate) or under conditions equivalent thereto.

1. HER2

HER2 is one of the oncogene products of a typical growth factor receptoroncogene identified as human epidermal cell growth factor receptor2-related oncogene, and is a transmembrane receptor protein having amolecular weight of 185 kDa and having a tyrosine kinase domain. HER2 isa member of the EGFR family consisting of HER1 (EGFR, ErbB-1), HER2(neu, ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4) and is known to beautophosphorylated at intracellular tyrosine residues by its homodimerformation or heterodimer formation with another EGFR receptor HER1,HER3, or HER4 and is itself activated in that manner, thereby playing animportant role in cell growth, differentiation, and survival in normalcells and tumor cells.

As for the HER2 protein to be used in the present invention, the HER2protein can be directly purified from HER2-expressing cells of a humanor a non-human mammal (such as a rat or a mouse) and used, or a cellmembrane fraction of the above-described cells can be prepared and used.Further, HER2 can be obtained by in vitro synthesis thereof orproduction thereof in a host cell through genetic engineering. In thegenetic engineering, specifically, after HER2 cDNA is integrated into avector capable of expressing HER2 cDNA, the HER2 protein can be obtainedby synthesizing it in a solution containing an enzyme, a substrate andan energy substance required for transcription and translation, or byexpressing HER2 in another prokaryotic or eucaryotic transformed hostcell. Alternatively, the above-described genetically engineeredHER2-expressing cells, or a cell line expressing HER2 may be used as theHER2 protein.

The DNA sequence and amino acid sequence of HER2 are disclosed on apublic database, and can be referred to, for example, under AccessionNo. M11730 (GenBank), NP_004439.2 (NCBI), or the like.

Further, a protein which consists of an amino acid sequence wherein oneor several amino acids are substituted, deleted and/or added in any ofthe above-described amino acid sequences of HER2 and also has abiological activity equivalent to that of the protein is also includedin HER2.

Human HER2 protein is composed of a signal sequence consisting ofN-terminal 22 amino acid residues, an extracellular domain consisting of630 amino acid residues, a transmembrane domain consisting of 23 aminoacid residues, and an intracellular domain consisting of 580 amino acidresidues.

2. Production of Anti-HER2 Antibody

The antibody against HER2 of the present invention can be obtainedaccording to, for example, a method usually carried out in the art,which involves immunizing animals with HER2 or an arbitrary polypeptideselected from the amino acid sequence of HER2 and collecting andpurifying antibodies produced in vivo. The biological species of HER2 tobe used as an antigen is not limited to being human, and an animal canbe immunized with HER2 derived from an animal other than humans such asa mouse or a rat or with rat p185neu. In this case, by examining thecross-reactivity between an antibody binding to the obtainedheterologous HER2 and human HER2, an antibody applicable to a humandisease can be selected.

Further, a monoclonal antibody can be obtained from a hybridomaestablished by fusing antibody-producing cells which produce an antibodyagainst HER2 with myeloma cells according to a known method (forexample, Kohler and Milstein, Nature, (1975) 256, pp. 495-497; Kennet,R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, N.Y. (1980)).

HER2 to be used as an antigen can be obtained by expressing HER2 gene ina host cell using genetic engineering.

Specifically, a vector capable of expressing HER2 gene is produced, andthe resulting vector is transfected into a host cell to express thegene, and then, the expressed HER2 is purified.

Alternatively, the above-described genetically engineeredHER2-expressing cells, or a cell line expressing HER2 may be used as theHER2 protein. The anti-HER2 antibody can be obtained by a procedureknown in the art. Hereinafter, a method of obtaining an antibody againstHER2 is specifically described.

(1) Preparation of Antigen

Examples of the antigen to be used for producing the anti-HER2 antibodyinclude HER2, or a polypeptide consisting of a partial amino acidsequence comprising at least 6 consecutive amino acids of HER2, or aderivative obtained by adding a given amino acid sequence or carrierthereto.

HER2 can be purified directly from human tumor tissues or tumor cellsand used. Further, HER2 can be obtained by synthesizing it in vitro orby producing it in a host cell by genetic engineering.

With respect to the genetic engineering, specifically, after HER2 cDNAis integrated into a vector capable of expressing HER2 cDNA, HER2 can beobtained by synthesizing it in a solution containing an enzyme, asubstrate and an energy substance required for transcription andtranslation, or by expressing HER2 in another prokaryotic or eucaryotictransformed host cell.

Further, the antigen can also be obtained as a secretory protein byexpressing a fusion protein obtained by ligating the extracellulardomain of HER2, which is a membrane protein, to the constant region ofan antibody in an appropriate host-vector system.

HER2 cDNA can be obtained by, for example, a so-called PCR method inwhich a polymerase chain reaction is performed using a cDNA libraryexpressing HER2 cDNA as a template and primers which specificallyamplify HER2 cDNA (PCR; Saiki, R. K., et al., Science, (1988) 239, pp.487-489).

As the in vitro synthesis of the polypeptide, for example, RapidTranslation System (RTS) manufactured by Roche Diagnostics, Inc. can beexemplified, but it is not limited thereto.

Examples of the prokaryotic host cells include Escherichia coli andBacillus subtilis. In order to transform the host cells with a targetgene, the host cells are transformed by a plasmid vector comprising areplicon, i.e., a replication origin derived from a species compatiblewith the host, and a regulatory sequence. Further, the vector preferablyhas a sequence capable of imposing phenotypic selectivity on thetransformed cell.

Examples of the eucaryotic host cells include vertebrate cells, insectcells, and yeast cells. As the vertebrate cells, for example, simian COScells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650; ATCC:American Type Culture Collection), murine fibroblasts NIH3T3 (ATCC No.CRL-1658), and dihydrofolate reductase-deficient strains (Urlaub, G. andChasin, L. A., Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220) ofChinese hamster ovarian cells (CHO cells; ATCC: CCL-61); and the likeare often used, however, the cells are not limited thereto.

The thus obtained transformant can be cultured according to a methodusually carried out in the art, and by the culturing of thetransformant, a target polypeptide is produced intracellularly orextracellularly.

A suitable medium to be used for the culturing can be selected fromvarious commonly used culture media depending on the employed hostcells. If Escherichia coli is employed, for example, an LB mediumsupplemented with an antibiotic such as ampicillin or IPMG as needed canbe used.

A recombinant protein produced intracellularly or extracellularly by thetransformant through such culturing can be separated and purified by anyof various known separation methods utilizing the physical or chemicalproperty of the protein.

Specific examples of the methods include treatment with a common proteinprecipitant, ultrafiltration, various types of liquid chromatographysuch as molecular sieve chromatography (gel filtration), adsorptionchromatography, ion exchange chromatography, and affinitychromatography, dialysis, and a combination thereof.

Further, by attaching a tag of six histidine residues to a recombinantprotein to be expressed, the protein can be efficiently purified with anickel affinity column. Alternatively, by attaching the IgG Fc region toa recombinant protein to be expressed, the protein can be efficientlypurified with a protein A column.

By combining the above-described methods, a large amount of a targetpolypeptide can be easily produced in high yield and high purity.

The above-described transformant itself may be used as the antigen. Acell line expressing HER2 may also be used as the antigen. Examples ofsuch a cell line can include human breast cancer lines SK-BR-3, BT-474,KPL-4, and JIMT-1, a human gastric cancer line NCI-N87, and a humanovarian cancer line SK-OV-3. The cell line of the present invention isnot limited to these cell lines as long as it expresses HER2.

(2) Production of Anti-HER2 Monoclonal Antibody

Examples of the antibody specifically bind to HER2 include a monoclonalantibody specifically bind to HER2, and a method of obtaining suchantibody is as described below.

The production of a monoclonal antibody generally requires the followingoperational steps of:

(a) purifying a biopolymer to be used as an antigen, or preparingantigen-expressing cells;

(b) preparing antibody-producing cells by immunizing an animal byinjection of the antigen, collecting the blood, assaying its antibodytiter to determine when the spleen is excised;

(c) preparing myeloma cells (hereinafter referred to as “myeloma”);

(d) fusing the antibody-producing cells with the myeloma;

(e) screening a group of hybridomas producing a desired antibody;

(f) dividing the hybridomas into single cell clones (cloning);

(g) optionally, culturing the hybridoma or rearing an animal implantedwith the hybridoma for producing a large amount of monoclonal antibody;

(h) examining the thus produced monoclonal antibody for biologicalactivity and binding specificity, or assaying the same for properties asa labeled reagent; and the like.

Hereinafter, the method of producing a monoclonal antibody will bedescribed in detail following the above steps, however, the method isnot limited thereto, and, for example, antibody-producing cells otherthan spleen cells and myeloma can be used.

(a) Purification of Antigen

As the antigen, HER2 prepared by the method as described above or apartial peptide thereof can be used.

Further, a membrane fraction prepared from recombinant cells expressingHER2 or the recombinant cells expressing HER2 themselves, and also apartial peptide of the protein of the invention chemically synthesizedby a method known to those skilled in the art can also be used as theantigen.

Furthermore, a HER2-expressing cell line can also be used as theantigen.

(b) Preparation of Antibody-Producing Cells

The antigen obtained in the step (a) is mixed with an adjuvant such asFreund's complete or incomplete adjuvant or auxiliary agent such asaluminum potassium sulfate and the resulting mixture is used as animmunogen to immunize an experimental animal. Another method involvesimmunizing an experimental animal with antigen-expressing cells as animmunogen. As the experimental animal, any animal used in a knownhybridoma production method can be used without hindrance. Specifically,for example, a mouse, a rat, a goat, sheep, cattle, a horse, or the likecan be used. However, from the viewpoint of ease of availability ofmyeloma cells to be fused with the extracted antibody-producing cells, amouse or a rat is preferably used as the animal to be immunized.

Further, the strain of a mouse or a rat to be used is not particularlylimited, and in the case of a mouse, for example, various strains suchas A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH,HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, and 129 and the like can beused, and in the case of a rat, for example, Wistar, Low, Lewis,Sprague, Dawley, ACI, BN, Fischer and the like can be used.

These mice and rats are commercially available frombreeders/distributors of experimental animals, for example, CLEA Japan,Inc. and Charles River Laboratories Japan, Inc.

As the animal to be immunized, in consideration of compatibility offusing with myeloma cells described below, in the case of a mouse,BALB/c strain, and in the case of a rat, Wistar and Low strains areparticularly preferred.

Further, in consideration of antigenic homology between humans and mice,it is also preferred to use a mouse having decreased biological functionto remove auto-antibodies, that is, a mouse with an autoimmune disease.

The age of such mouse or rat at the time of immunization is preferably 5to 12 weeks of age, more preferably 6 to 8 weeks of age.

In order to immunize an animal with HER2 or a recombinant thereof, forexample, a known method described in detail in, for example, Weir, D.M., Handbook of Experimental Immunology Vol. I. II. III., BlackwellScientific Publications, Oxford (1987); Kabat, E. A. and Mayer, M. M.,Experimental Immunochemistry, Charles C Thomas Publisher Springfield,Ill. (1964) or the like can be used.

Among these immunization methods, a preferred specific method in thepresent invention is, for example, as follows.

That is, first, a membrane protein fraction serving as the antigen orcells caused to express the antigen is/are intradermally orintraperitoneally administrated to an animal. However, the combinationof both routes of administration is preferred for increasing theimmunization efficiency, and when intradermal administration isperformed in the first half and intraperitoneal administration isperformed in the latter half or only at the last dosing, theimmunization efficiency can be particularly increased.

The administration schedule of the antigen varies depending on the typeof animal to be immunized, individual difference or the like. However,in general, an administration schedule in which the frequency ofadministration of the antigen is 3 to 6 times and the dosing interval is2 to 6 weeks is preferred, and an administration schedule in which thefrequency of administration of the antigen is 3 to 4 times and thedosing interval is 2 to 4 weeks is more preferred.

Further, the dose of the antigen varies depending on the type of animal,individual differences or the like, however, the dose is generally setto 0.05 to 5 mg, preferably about 0.1 to 0.5 mg.

A booster immunization is performed 1 to 6 weeks, preferably 1 to 4weeks, more preferably 1 to 3 weeks after the administration of theantigen as described above. When the immunogen is cells, 1×10⁶ to 1×10′cells are used.

The dose of the antigen at the time of performing the boosterimmunization varies depending on the type or size of animal or the like,however, in the case of, for example, a mouse, the dose is generally setto 0.05 to 5 mg, preferably 0.1 to 0.5 mg, more preferably about 0.1 to0.2 mg. When the immunogen is cells, 1×10⁶ to 1×10⁷ cells are used.

Spleen cells or lymphocytes including antibody-producing cells areaseptically removed from the immunized animal after 1 to 10 days,preferably 2 to 5 days, more preferably 2 to 3 days from the boosterimmunization. At this time, the antibody titer is measured, and if ananimal having a sufficiently increased antibody titer is used as asupply source of the antibody-producing cells, the subsequent procedurecan be carried out more efficiently.

Examples of the method of measuring the antibody titer to be used hereinclude an RIA method and an ELISA method, but the method is not limitedthereto. For example, if an ELISA method is employed, the measurement ofthe antibody titer in the invention can be carried out according to theprocedures as described below.

First, a purified or partially purified antigen is adsorbed to thesurface of a solid phase such as a 96-well plate for ELISA, and thesurface of the solid phase having no antigen adsorbed thereto is coveredwith a protein unrelated to the antigen such as bovine serum albumin(BSA). After washing the surface, the surface is brought into contactwith a serially-diluted sample (for example, mouse serum) as a primaryantibody to allow the antibody in the sample to bind to the antigen.

Further, as a secondary antibody, an antibody labeled with an enzymeagainst a mouse antibody is added and is allowed to bind to the mouseantibody. After washing, a substrate for the enzyme is added and achange in absorbance which occurs due to color development induced bydegradation of the substrate or the like is measured and the antibodytiter is calculated based on the measurement.

The separation of the antibody-producing cells from the spleen cells orlymphocytes of the immunized animal can be carried out according to aknown method (for example, Kohler et al., Nature (1975), 256, p. 495;Kohler et al., Eur. J. Immunol. (1977), 6, p. 511; Milstein et al.,Nature (1977), 266, p. 550; Walsh, Nature (1977), 266, p. 495). Forexample, in the case of spleen cells, a general method in which theantibody-producing cells are separated by homogenizing the spleen toobtain the cells through filtration with a stainless steel mesh andsuspending the cells in Eagle's Minimum Essential Medium (MEM) can beemployed.

(c) Preparation of Myeloma Cells (Hereinafter Referred to as “Myeloma”)

The myeloma cells to be used for cell fusion are not particularlylimited and suitable cells can be selected from known cell lines.However, in consideration of convenience when a hybridoma is selectedfrom fused cells, it is preferred to use an HGPRT (hypoxanthine-guaninephosphoribosyl transferase) deficient strain whose selection procedurehas been established.

More specifically, examples of the HGPRT-deficient strain includeX63-Ag8(X63), NS1-ANS/1(NS1), P3X63-Ag8.U1(P3U1), X63-Ag8.653(X63.653),SP2/0-Agl4(SP2/0), MPC11-45.6TG1.7(45.6TG), FO, S149/5XXO, and BU.1derived from mice; 210.RSY3.Ag.1.2.3(Y3) derived from rats; andU266AR(SKO-007), GM1500.GTG-A12(GM1500), UC729-6, LICR-LOW-HMy2(HMy2)and 8226AR/NIP4-1(NP41) derived from humans. These HGPRT-deficientstrains are available from, for example, ATCC or the like.

These cell strains are subcultured in an appropriate medium such as an8-azaguanine medium [a medium obtained by adding 8-azaguanine to an RPMI1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin,and fetal calf serum (hereinafter referred to as “FCS”)], Iscove'sModified Dulbecco's Medium (hereinafter referred to as “IMDM”), orDulbecco's Modified Eagle Medium (hereinafter referred to as “DMEM”). Inthis case, 3 to 4 days before performing cell fusion, the cells aresubcultured in a normal medium (for example, an ASF104 medium(manufactured by Ajinomoto Co., Ltd.) containing 10% FCS) to ensure notless than 2×10⁷ cells on the day of cell fusion.

(d) Cell Fusion

Fusion between the antibody-producing cells and the myeloma cells can beappropriately performed according to a known method (Weir, D. M.Handbook of Experimental Immunology Vol. I. II. III., BlackwellScientific Publications, Oxford (1987); Kabat, E. A. and Mayer, M. M.,Experimental Immunochemistry, Charles C Thomas Publisher, Springfield,Ill. (1964), etc.), under conditions such that the survival rate ofcells is not excessively reduced.

As such a method, for example, a chemical method in which theantibody-producing cells and the myeloma cells are mixed in a solutioncontaining a polymer such as polyethylene glycol at a highconcentration, a physical method using electric stimulation, or the likecan be used. Among these methods, a specific example of the chemicalmethod is as described below.

That is, in the case where polyethylene glycol is used in the solutioncontaining a polymer at a high concentration, the antibody-producingcells and the myeloma cells are mixed in a solution of polyethyleneglycol having a molecular weight of 1500 to 6000, more preferably 2000to 4000 at a temperature of from 30 to 40° C., preferably from 35 to 38°C. for 1 to 10 minutes, preferably 5 to 8 minutes.

(e) Selection of a Group of Hybridomas

The method of selecting hybridomas obtained by the above-described cellfusion is not particularly limited. Usually, an HAT (hypoxanthine,aminopterin, thymidine) selection method (Kohler et al., Nature (1975),256, p. 495; Milstein et al., Nature (1977), 266, p. 550) is used.

This method is effective when hybridomas are obtained using the myelomacells of an HGPRT-deficient strain which cannot survive in the presenceof aminopterin. That is, by culturing unfused cells and hybridomas in anHAT medium, only hybridomas resistant to aminopterin are selectivelyallowed to survive and proliferate.

(f) Division into Single Cell Clone (Cloning)

As a cloning method for hybridomas, a known method such as amethylcellulose method, a soft agarose method, or a limiting dilutionmethod can be used (see, for example, Barbara, B. M. and Stanley, M. S.:Selected Methods in Cellular Immunology, W. H. Freeman and Company, SanFrancisco (1980)). Among these methods, particularly, athree-dimensional culture method such as a methylcellulose method ispreferred. For example, the group of hybridomas produced by cell fusionare suspended in a methylcellulose medium such as ClonaCell-HY SelectionMedium D (manufactured by StemCell Technologies, Inc., #03804) andcultured. Then, the formed hybridoma colonies are collected, wherebymonoclonal hybridomas can be obtained. The collected respectivehybridoma colonies are cultured, and a hybridoma which has beenconfirmed to have a stable antibody titer in an obtained hybridomaculture supernatant is selected as a HER2 monoclonal antibody-producinghybridoma strain.

(g) Preparation of Monoclonal Antibody by Culturing Hybridoma

By culturing the thus selected hybridoma, a monoclonal antibody can beefficiently obtained. However, prior to culturing, it is preferred toperform screening of a hybridoma which produces a target monoclonalantibody.

In such screening, a known method can be employed.

The measurement of the antibody titer in the invention can be carriedout by, for example, an ELISA method explained in item (b) describedabove.

The hybridoma obtained by the method described above can be stored in afrozen state in liquid nitrogen or in a freezer at −80° C. or below.

After completion of cloning, the medium is changed from an HT medium toa normal medium, and the hybridoma is cultured.

Large-scale culture is performed by rotation culture using a largeculture bottle or by spinner culture. From the supernatant obtained bythe large-scale culture, a monoclonal antibody which specifically bindsto the protein of the invention can be obtained by purification using amethod known to those skilled in the art such as gel filtration.

Further, the hybridoma is injected into the abdominal cavity of a mouseof the same strain as the hybridoma (for example, the above-describedBALB/c) or a Nu/Nu mouse to proliferate the hybridoma, whereby theascites containing a large amount of the monoclonal antibody of theinvention can be obtained.

In the case where the hybridoma is administrated in the abdominalcavity, if a mineral oil such as 2,6,10,14-tetramethyl pentadecane(pristane) is administrated 3 to 7 days prior thereto, a larger amountof the ascites can be obtained.

For example, an immunosuppressant is previously injected into theabdominal cavity of a mouse of the same strain as the hybridoma toinactivate T cells. 20 days thereafter, 10⁶ to 10⁷ hybridoma clone cellsare suspended in a serum-free medium (0.5 ml), and the suspension isadministrated in the abdominal cavity of the mouse. In general, when theabdomen is expanded and filled with the ascites, the ascites iscollected from the mouse. By this method, the monoclonal antibody can beobtained at a concentration which is about 100 times or much higher thanthat in the culture solution.

The monoclonal antibody obtained by the above-described method can bepurified by a method described in, for example, Weir, D. M.: Handbook ofExperimental Immunology Vol. I, II, III, Blackwell ScientificPublications, Oxford (1978).

The thus obtained monoclonal antibody has high antigen specificity forHER2. Examples of the monoclonal antibody of the present invention caninclude, but are not particularly limited to, a mouse monoclonalantibody 4D5 (ATCC CRL 10463).

(h) Assay of Monoclonal Antibody

The isotype and subclass of the thus obtained monoclonal antibody can bedetermined as follows.

First, examples of the identification method include an Ouchterlonymethod, an ELISA method, and an RIA method.

An Ouchterlony method is simple, but when the concentration of themonoclonal antibody is low, a condensation operation is required.

On the other hand, when an ELISA method or an RIA method is used, bydirectly reacting the culture supernatant with an antigen-adsorbed solidphase and using antibodies corresponding to various types ofimmunoglobulin isotypes and subclasses as secondary antibodies, theisotype and subclass of the monoclonal antibody can be identified.

In addition, as a simpler method, a commercially availableidentification kit (for example, Mouse Typer Kit manufactured by Bio-RadLaboratories, Inc.) or the like can also be used.

Further, the quantitative determination of a protein can be performed bythe Folin Lowry method and a method of calculation based on theabsorbance at 280 nm (1.4 (OD 280)=Immunoglobulin 1 mg/ml).

Further, even when the monoclonal antibody is separately andindependently obtained by performing again the steps of (a) to (h) in(2), it is possible to obtain an antibody having a cytotoxic activityequivalent to that of the HER2 antibody obtained in the the step of (g).As one example of such an antibody, an antibody which binds to the sameepitope as the HER2 antibody obtained in the step of (g) can beexemplified. If a newly produced monoclonal antibody binds to a partialpeptide or a partial tertiary structure to which the anti-HER2 antibodybinds, it can be determined that the monoclonal antibody binds to thesame epitope as the anti-HER2 antibody. Further, by confirming that themonoclonal antibody competes with the anti-HER2 antibody for the bindingto HER2 (that is, the monoclonal antibody inhibits the binding betweenthe anti-HER2 antibody and HER2), it can be determined that themonoclonal antibody binds to the same epitope as the anti-HER2 antibodyeven if the specific epitope sequence or structure has not beendetermined. When it is confirmed that the monoclonal antibody binds tothe same epitope as the anti-HER2 antibody, the monoclonal antibody isstrongly expected to have an antigen-binding affinity or biologicalactivity equivalent to that of the anti-HER2 antibody.

(3) Other Antibodies

The antibody of the invention includes not only the above-describedmonoclonal antibody against HER2 but also a recombinant antibodyobtained by artificial modification for the purpose of decreasingheterologous antigenicity to humans such as a chimeric antibody, ahumanized antibody and a human antibody. These antibodies can beproduced using a known method.

As the chimeric antibody, an antibody in which antibody variable andconstant regions are derived from different species, for example, achimeric antibody in which a mouse- or rat-derived antibody variableregion is connected to a human-derived antibody constant region can beexemplified (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).Examples of the chimeric antibody of the present invention can include,but are not particularly limited to, a chimeric antibody 4D5 comprisinga heavy chain constant region of human IgG1 or IgG2.

As the humanized antibody, an antibody obtained by integrating only acomplementarity determining region (CDR) into a human-derived antibody(see Nature (1986) 321, pp. 522-525), and an antibody obtained bygrafting a part of the amino acid residues of the framework as well asthe CDR sequence to a human antibody by a CDR-grafting method (WO90/07861), and an antibody humanized using gene conversion mutagenesisstrategy (U.S. Pat. No. 5,821,337) can be exemplified.

The term “several” as used herein refers to 1 to 10, 1 to 9, 1 to 8, 1to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

As the amino acid substitution in this specification, a conservativeamino acid substitution is preferred. The conservative amino acidsubstitution refers to a substitution occurring within a group of aminoacids related to amino acid side chains. Preferred amino acid groups areas follows: an acidic group (aspartic acid and glutamic acid); a basicgroup (lysine, arginine, and histidine); a non-polar group (alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine, andtryptophan); and an uncharged polar family (glycine, asparagine,glutamine, cysteine, serine, threonine, and tyrosine). More preferredamino acid groups are as follows: an aliphatic hydroxy group (serine andthreonine); an amide-containing group (asparagine and glutamine); analiphatic group (alanine, valine, leucine, and isoleucine); and anaromatic group (phenylalanine, tryptophan, and tyrosine). Such an aminoacid substitution is preferably performed within a range which does notimpair the properties of a substance having the original amino acidsequence.

By combining a sequence having a high homology with the above-describedheavy chain amino acid sequence with a sequence having a high homologywith the above-described light chain amino acid sequence, it is possibleto select an antibody having a biological activity equivalent to that ofeach of the above-described antibodies. Such a homology is generally ahomology of 80% or more, preferably a homology of 90% or more, morepreferably a homology of 95% or more, most preferably a homology of 99%or more. Further, by combining an amino acid sequence wherein one toseveral amino acid residues are substituted, deleted or added in theheavy chain or light chain amino acid sequence, it is also possible toselect an antibody having a biological activity equivalent to that ofeach of the above-described antibodies. The term “homology” as usedherein is used with the same meaning as “identity”.

The homology between two amino acid sequences can be determined usingdefault parameters of Blast algorithm version 2.2.2 (Altschul, StephenF., Thomas L. Madden, Alejandro A. Schaeffer, Jinghui Zhang, ZhengZhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25: 3389-3402). The Blast algorithm can be used alsothrough the Internet by accessing the site www.ncbi.nlm.nih.gov/blast.

Further, the antibody of the invention includes a human antibody whichbinds to HER2. An anti-HER2 human antibody refers to a human antibodyhaving only a sequence of an antibody derived from a human chromosome.The anti-HER2 human antibody can be obtained by a method using a humanantibody-producing mouse having a human chromosome fragment comprisingheavy and light chain genes of a human antibody (see Tomizuka, K. etal., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl.Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal CellTechnology: Basic and Applied Aspects vol. 10, pp. 69-73 (Kitagawa, Y.,Matuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999;Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727,etc.).

Such a human antibody-producing mouse can be created specifically asfollows. A genetically modified animal in which endogenousimmunoglobulin heavy and light chain gene loci have been disrupted, andinstead, human immunoglobulin heavy and light chain gene loci have beenintroduced via a yeast artificial chromosome (YAC) vector or the like iscreated by producing a knockout animal and a transgenic animal andmating these animals.

Further, according to a recombinant DNA technique, by using cDNAsencoding each of such a heavy chain and a light chain of a humanantibody, and preferably a vector comprising such cDNAs, eukaryoticcells are transformed, and a transformant cell which produces arecombinant human monoclonal antibody is cultured, whereby the antibodycan also be obtained from the culture supernatant.

Here, as the host, for example, eukaryotic cells, preferably mammaliancells such as CHO cells, lymphocytes, or myeloma cells can be used.

Further, a method of obtaining a phage display-derived human antibodyselected from a human antibody library (see Wormstone, I. M. et al.,Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp.2301-2308; Carmen, S. et al., Briefings in Functional Genomics andProteomics (2002), 1 (2), pp. 189-203; Siriwardena, D. et al.,Ophthalmology (2002) 109 (3), pp. 427-431, etc.) is also known.

For example, a phage display method in which a variable region of ahuman antibody is expressed on the surface of a phage as a single-chainantibody (scFv), and a phage which binds to an antigen is selected(Nature Biotechnology (2005), 23, (9), pp. 1105-1116) can be used.

By analyzing the gene of the phage selected based on the binding to anantigen, a DNA sequence encoding the variable region of a human antibodywhich binds to an antigen can be determined.

If the DNA sequence of scFv which binds to an antigen is determined, ahuman antibody can be obtained by preparing an expression vectorcomprising the sequence and introducing the vector into an appropriatehost to express it (WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236,WO 93/19172, WO 95/01438, WO 95/15388; Annu. Rev. Immunol. (1994) 12,pp. 433-455, Nature Biotechnology (2005) 23 (9), pp. 1105-1116).

As one example of another index for use in the comparison of theproperties of antibodies, the stability of antibodies can beexemplified. The differential scanning calorimetry (DSC) is a devicecapable of quickly and accurately measuring a thermal denaturationmidpoint temperature (Tm) to be used as a favorable index of therelative conformational stability of proteins. By measuring the Tmvalues using DSC and comparing the values, a difference in thermalstability can be compared. It is known that the storage stability ofantibodies shows some correlation with the thermal stability ofantibodies (Lori Burton, et. al., Pharmaceutical Development andTechnology (2007) 12, pp. 265-273), and a preferred antibody can beselected by using thermal stability as an index. Examples of otherindices for selecting antibodies include the following features: theyield in an appropriate host cell is high; and the aggregability in anaqueous solution is low. For example, an antibody which shows thehighest yield does not always show the highest thermal stability, andtherefore, it is necessary to select an antibody most suitable for theadministration to humans by making comprehensive evaluation based on theabove-described indices.

In the present invention, a modified variant of the antibody is alsoincluded. The modified variant refers to a variant obtained bysubjecting the antibody of the present invention to chemical orbiological modification. Examples of the chemically modified variantinclude variants chemically modified by linking a chemical moiety to anamino acid skeleton, variants chemically modified with an N-linked orO-linked carbohydrate chain, etc. Examples of the biologically modifiedvariant include variants obtained by post-translational modification(such as N-linked or O-linked glycosylation, N- or C-terminalprocessing, deamidation, isomerization of aspartic acid, or oxidation ofmethionine), and variants in which a methionine residue has been addedto the N terminus by being expressed in a prokaryotic host cell.Further, an antibody labeled so as to enable the detection or isolationof the antibody or an antigen of the invention, for example, anenzyme-labeled antibody, a fluorescence-labeled antibody, and anaffinity-labeled antibody are also included in the meaning of themodified variant. Such a modified variant of the antibody of theinvention is useful for improving the stability and blood retention ofthe antibody, reducing the antigenicity thereof, detecting or isolatingan antibody or an antigen, and so on.

Further, by regulating the modification of a glycan which is linked tothe antibody of the invention (glycosylation, defucosylation, etc.), itis possible to enhance an antibody-dependent cellular cytotoxicactivity. As the technique for regulating the modification of a glycanof antibodies, WO 99/54342, WO 00/61739, WO 02/31140, etc. are known.However, the technique is not limited thereto. In the antibody of thepresent invention, an antibody in which the modification of a glycan isregulated is also included.

In the case where an antibody is produced by first isolating an antibodygene and then introducing the gene into an appropriate host, acombination of an appropriate host and an appropriate expression vectorcan be used. Specific examples of the antibody gene include acombination of a gene encoding a heavy chain sequence of an antibodydescribed in this specification and a gene encoding a light chainsequence thereof. When a host cell is transformed, it is possible toinsert the heavy chain sequence gene and the light chain sequence geneinto the same expression vector, and also into different expressionvectors separately.

In the case where eukaryotic cells are used as the host, animal cells,plant cells, and eukaryotic microorganisms can be used. As the animalcells, mammalian cells, for example, simian COS cells (Gluzman, Y.,Cell, (1981) 23, pp. 175-182, ATCC CRL-1650), murine fibroblasts NIH3T3(ATCC No. CRL-1658), and dihydrofolate reductase-deficient strains(Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA (1980) 77, pp.4126-4220) of Chinese hamster ovarian cells (CHO cells; ATCC: CCL-61)can be exemplified.

In the case where prokaryotic cells are used, for example, Escherichiacoli and Bacillus subtilis can be exemplified.

By introducing a desired antibody gene into these cells throughtransformation, and culturing the thus transformed cells in vitro, theantibody can be obtained. In the above-described culture method, theyield may sometimes vary depending on the sequence of the antibody, andtherefore, it is possible to select an antibody which is easily producedas a pharmaceutical by using the yield as an index among the antibodieshaving an equivalent binding activity. Therefore, in the antibody of thepresent invention, an antibody obtained by a method of producing anantibody, characterized by including a step of culturing the transformedhost cell and a step of collecting a desired antibody from a culturedproduct obtained in the culturing step is also included.

It is known that a lysine residue at the carboxyl terminus of the heavychain of an antibody produced in a cultured mammalian cell is deleted(Journal of Chromatography A, 705: 129-134 (1995)), and it is also knownthat two amino acid residues (glycine and lysine) at the carboxylterminus of the heavy chain of an antibody produced in a culturedmammalian cell are deleted and a proline residue newly located at thecarboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83(2007)). However, such deletion and modification of the heavy chainsequence do not affect the antigen-binding affinity and the effectorfunction (the activation of a complement, the antibody-dependentcellular cytotoxicity, etc.) of the antibody. Therefore, in the antibodyaccording to the present invention, an antibody subjected to suchmodification and a functional fragment of the antibody is also included,and a deletion variant in which one or two amino acids have been deletedat the carboxyl terminus of the heavy chain, a variant obtained byamidation of the deletion variant (for example, a heavy chain in whichthe carboxyl terminal proline residue has been amidated), and the likeare also included. The type of deletion variant having a deletion at thecarboxyl terminus of the heavy chain of the antibody according to theinvention is not limited to the above variants as long as theantigen-binding affinity and the effector function are conserved. Thetwo heavy chains constituting the antibody according to the inventionmay be of one type selected from the group consisting of a full-lengthheavy chain and the above-described deletion variant, or may be of twotypes in combination selected therefrom. The ratio of the amount of eachdeletion variant can be affected by the type of cultured mammalian cellswhich produce the antibody according to the invention and the cultureconditions, however, a case where one amino acid residue at the carboxylterminus has been deleted in both of the two heavy chains contained asmain components in the antibody according to the invention can beexemplified.

As isotype of the antibody of the invention, for example, IgG (IgG1,IgG2, IgG3, IgG4) can be exemplified, and IgG1 or IgG2 can beexemplified preferably.

As the biological activity of the antibody, generally an antigen-bindingactivity, an activity of internalizing in cells expressing an antigen bybinding to the antigen, an activity of neutralizing the activity of anantigen, an activity of enhancing the activity of an antigen, anantibody-dependent cellular cytotoxicity (ADCC) activity, acomplement-dependent cytotoxicity (CDC) activity, and anantibody-dependent cell-mediated phagocytosis (ADCP) can be exemplified.The biological activity of the antibody of the present invention is abinding activity to HER2, and preferably an activity of internalizing inHER2-expressing cells by binding to HER2. Further, the antibody of thepresent invention may have an ADCC activity, a CDC activity, and/or anADCP activity in addition to an activity of internalizing in cells.

The obtained antibody can be purified to homogeneity. The separation andpurification of the antibody may be performed employing a conventionalprotein separation and purification method. For example, the antibodycan be separated and purified by appropriately selecting and combiningcolumn chromatography, filter filtration, ultrafiltration, saltprecipitation, dialysis, preparative polyacrylamide gel electrophoresis,isoelectric focusing electrophoresis, and the like (Strategies forProtein Purification and Characterization: A Laboratory Course Manual,Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press(1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, ColdSpring Harbor Laboratory (1988)), but the method is not limited thereto.

Examples of such chromatography include affinity chromatography, ionexchange chromatography, hydrophobic chromatography, gel filtrationchromatography, reverse phase chromatography, and adsorptionchromatography.

Such chromatography can be performed employing liquid chromatographysuch as HPLC or FPLC.

As a column to be used in affinity chromatography, a Protein A columnand a Protein G column can be exemplified. For example, as a columnusing a Protein A column, Hyper D, POROS, Sepharose FF (PharmaciaCorporation) and the like can be exemplified.

Further, by using a carrier having an antigen immobilized thereon, theantibody can also be purified utilizing the binding property of theantibody to the antigen.

[Antitumor Compound]

The antitumor compound to be conjugated to the anti-HER2 antibody-drugconjugate of the present invention is explained. The antitumor compoundused in the present invention is not particularly limited provided it isa compound having an antitumor effect and a substituent group or apartial structure allowing connection to a linker structure. When a partor whole linker is cleaved in tumor cells, the antitumor compound moietyis released to exhibit the antitumor effect of the antitumor compound.As the linker is cleaved at a connecting position to the drug, theantitumor compound is released in an unmodified structure to exhibit itsintrinsic antitumor effect.

As the antitumor compound used in the present invention, exatecan(((1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione;shown in the following formula), one of the camptothecin derivatives,can be preferably used.

Although having an excellent antitumor effect, exatecan has not beencommercialized as an antitumor drug. The compound can be easily obtainedby a known method and the amino group at position 1 can be preferablyused as the connecting position to the linker structure. Further,although exatecan can be also released in tumor cells while part of thelinker is still attached thereto, it is an excellent compound exhibitingan excellent antitumor effect even in such a structure. Because exatecanhas a camptothecin structure, it is known that the equilibrium shifts toa structure with a closed lactone ring (closed ring) in an aqueousacidic medium (for example, pH 3 or so) but it shifts to a structurewith an open lactone ring (open ring) in an aqueous basic medium (forexample, pH 10 or so). A drug conjugate being introduced with anexatecan residue corresponding to the closed ring structure and the openring structure is also expected to have the same antitumor effect and itis needless to say that any of these structures is within the scope ofthe present invention.

Further examples of the antitumor compound can include doxorubicin,daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine,vinblastine, methotrexate, platinum-based antitumor agent (cisplatin orderivatives thereof), taxol or derivatives thereof, and othercamptothecins or derivatives thereof (antitumor agent described inJapanese Patent Laid-Open No. 6-87746).

With regard to the antibody-drug conjugate, the number of conjugateddrug molecules per antibody molecule is a key factor having an influenceon efficacy and safety. Production of the antibody-drug conjugate isperformed by defining the reaction conditions including the amounts ofraw materials and reagents used for the reaction so as to have aconstant number of conjugated drug molecules. A mixture containingdifferent numbers of conjugated drug molecules is generally obtainedunlike the chemical reaction of a low-molecular-weight compound. Thenumber of drugs conjugated in an antibody molecule is expressed orspecified by the average value, that is, the average number ofconjugated drug molecules. Unless specifically described otherwise as aprinciple, the number of conjugated drug molecules means the averagevalue except in the case in which it represents an antibody-drugconjugate having a specific number of conjugated drug molecules that isincluded in an antibody-drug conjugate mixture having different numbersof conjugated drug molecules.

The number of exatecan molecules conjugated to an antibody molecule iscontrollable, and as the average number of conjugated drug molecules perantibody molecule, about 1 to 10 exatecans can be connected. Preferably,it is 2 to 8, and more preferably 3 to 8. Meanwhile, a person skilled inthe art can design a reaction for conjugating a required number of drugmolecules to an antibody molecule based on the description of theExamples of the present application and can obtain an antibody-drugconjugate conjugated with a controlled number of exatecan molecules.

[Linker Structure]

With regard to the anti-HER2 antibody-drug conjugate of the presentinvention, the linker structure for conjugating an antitumor compound tothe anti-HER2 antibody is explained. The linker has a structure of thefollowing formula:

-L¹-L²-L^(P)-NH—(CH₂)n ¹-L^(a)-(CH₂)n ²—C(═O)—.

The antibody is connected to the terminal L¹ (terminal opposite to theconnection to L²), and the antitumor compound is connected to thecarbonyl group of the -L^(a)-(CH₂)n²—C(═O)— moiety.

n¹ represents an integer of 0 to 6 and is preferably an integer of 1 to5, and more preferably 1 to 3.

1. L¹

L¹ is represented by the following structure:

-(Succinimid-3-yl-N)—(CH₂)n ³—C(═O)—.

In the above, n³ is an integer of 2 to 8, “-(Succinimid-3-yl-N)—” has astructure represented by the following formula:

Position 3 of the above partial structure is the connecting position tothe anti-HER2 antibody. The bond to the antibody at position 3 ischaracterized by bonding with thioether formation. The nitrogen atom atposition 1 of the structure moiety is connected to the carbon atom ofthe methylene which is present within the linker including thestructure. Specifically, -(Succinimid-3-yl-N)— (CH₂)n³—C(═O)-L²- is astructure represented by the following formula (herein, “antibody-S—”originates from an antibody).

In the formula, n³ is an integer of 2 to 8, and preferably 2 to 5.

Specific examples of L¹ can include

-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—,

-(Succinimid-3-yl-N)—CH₂CH₂CH₂—C(═O)—,

-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂—C(═O)—, and

-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)—.

2. L²

L² is represented by the following structure:

—NH—(CH₂CH₂—O)n ⁴—CH₂CH₂—C(═O)—,

L² may not be present, and in such a case, L² is a single bond. n⁴ is aninteger of 1 to 6, and preferably 2 to 4. L² is connected to L¹ at itsterminal amino group and is connected to L^(P) at its carbonyl group atthe other terminal.

Specific examples of L² can include

—NH—CH₂CH₂—O—CH₂CH₂—C(═O)—,

—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)—,

—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)—,

—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)—,

—NH—CH₂CH₂—O—CH₂CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)—,

—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)—.

3. L^(P)

L^(P) is a peptide residue consisting of 2 to 7 amino acids.Specifically, it consists of an oligopeptide residue in which 2 to 7amino acids are linked by peptide bonding. L^(P) is connected to L² atits N terminus and is connected to the amino group of the —NH—(CH₂)n-L^(a)-(CH₂)n²—C(═O)— moiety of the linker at its C terminus. Here, theterm “peptide residue” or “oligopeptide residue” is a group derived froma peptide consisting of two or more amino acid residues and refers to adivalent group whose N terminus and C terminus are connecting positions.

The amino acid constituting L^(P) in the linker is not particularlylimited, however, examples thereof include an L- or a D-amino acid,preferably an L-amino acid. And, it can be an amino acid having astructure such as β-alanine, ε-aminocaproic acid, or γ-aminobutyric acidin addition to an α-amino acid, further, it can be a non-natural typeamino acid such as N-methylated amino acid.

The amino acid sequence of L^(P) is not particularly limited, butexamples of the constituting amino acids include phenylalanine (Phe; F),tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A),valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S),glutamic acid (Glu; E), and aspartic acid (Asp; D). Among them,preferred examples include phenylalanine, glycine, valine, lysine,citrulline, serine, glutamic acid, and aspartic acid. From these aminoacids, L^(P) having a sequence of amino acids optionally selected withor without overlaps may be constructed. Depending on the type of theamino acids, the drug release pattern can be controlled. The number ofamino acids can be between 2 to 7.

Specific examples of L^(P) can include

-GGF-, -DGGF-, -(D-)D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-,-GGFGG-, -DDGGFG-, -KDGGFG-, and -GGFGGGF-.

In the above, “(D-)D” represents a D-aspartic acid. Particularlypreferred examples of L^(P) for the antibody-drug conjugate of thepresent invention can include the tetrapeptide residue -GGFG-.4. L^(a)-(CH₂)n²—C(═O)—

L^(a) in L^(a)-(CH₂)n²—C(═O)— is a structure of —O— or a single bond. n²is an integer of 0 to 5, preferably 0 to 3, and more preferably 0 or 1.

Examples of L^(a)-(CH₂)n²—C(═O)— can include the following structures:

—O—CH₂—C(═O)—,

—O—CH₂CH₂—C(═O)—,—O—CH₂CH₂CH₂—C(═O)—,—O—CH₂CH₂CH₂CH₂—C(═O)—,—O—CH₂CH₂CH₂CH₂CH₂—C(═O)—,

—CH₂—C(═O)—,

—CH₂CH₂—C(═O)—,—CH₂CH₂CH₂—C(═O)—,—CH₂CH₂CH₂CH₂—C(═O)—,—CH₂CH₂CH₂CH₂CH₂—C(═O)—,

—O—C(═O)—.

Among these,

—O—CH₂—C(═O)—,

—O—CH₂CH₂—C(═O)—,

—O—C(═O)—,

or a case in which L^(a) is a single bond, and n² is 0 is preferred.

Specific examples of the structure represented by—NH—(CH₂)n¹-L^(a)-(CH₂)n²—C(═O)— of the linker can include

—NH—CH₂—C(═O)—,

—NH—CH₂CH₂—C(═O)—,—NH—CH₂—O—CH₂—C(═O)—,—NH—CH₂CH₂—O—CH₂—C(═O)—,—NH—CH₂CH₂CH₂—C(═O)—,—NH—CH₂CH₂CH₂CH₂—C(═O)—,—NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—,

—NH—CH₂—O—C(═O)—,

—NH—CH₂CH₂—O—C(═O)—,—NH—CH₂CH₂CH₂—O—C(═O)—,—NH—CH₂CH₂CH₂CH₂—O—C(═O)—.

Among these,

—NH—CH₂CH₂CH₂—C(═O)—,—NH—CH₂—O—CH₂—C(═O)—,—NH—CH₂CH₂—O—CH₂—C(═O)—are more preferred.

In the linker, the chain length of —NH—(CH₂)n¹-L^(a)(CH₂)n²—C(═O)— ispreferably a chain length of 4 to 7 atoms, and more preferably a chainlength of 5 or 6 atoms.

With regard to the anti-HER2 antibody-drug conjugate of the presentinvention, when it is transferred to the inside of tumor cells, it hasbeen suggested that the linker moiety is cleaved and the drug derivativehaving a structure represented byNH₂—(CH₂)n¹-L^(a)-(CH₂)n²—C(═O)—(NH-DX) is released to express anantitumor action. Examples of the antitumor derivative exhibiting anantitumor effect by releasing from the antibody-drug conjugate of thepresent invention include an antitumor derivative having a structuremoiety in which a terminal of the structure represented by —NH—(CH₂)n-L^(a)-(CH₂)n²—C(═O)— of the linker has an amino group, and thoseparticularly preferred include the following.

NH₂—CH₂CH₂—C(═O)—(NH-DX),NH₂—CH₂CH₂CH₂—C(═O)—(NH-DX),NH₂—CH₂—O—CH₂—C(═O)—(NH-DX),NH₂—CHCH₂—O—CH₂—C(═O)—(NH-DX).

Meanwhile, in case of NH₂—CH₂—O—CH₂—C(═O)—(NH-DX), it was confirmedthat, as the aminal structure in the molecule is unstable, it againundergoes a self-degradation to release the followingHO—CH₂—C(═O)—(NH-DX). Those compounds can be also preferably used as aproduction intermediate of the antibody-drug conjugate of the presentinvention.

For the antibody-drug conjugate of the present invention in whichexatecan is used as the drug, it is preferable that the drug-linkerstructure moiety [-L¹-L²-L^(P)-NH—(CH₂) n-L^(a)-(CH₂)n²—C(═O)—(NH-DX)]having the following structure is connected to an antibody. The averageconjugated number of said drug-linker structure moieties per antibodymolecule can be 1 to 10. Preferably, it is 2 to 8, and more preferably 3to 8.

-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CHCH₂₂—O—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX).

Among these, more preferred are the following.

-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂HH₂—O—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CHCH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX).

Particularly preferred are the following.

-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CHCH₂—C(═O)—(NH-DX)

With regard to the linker structure for conjugating the anti-HER2antibody and the drug in the antibody-drug conjugate of the presentinvention, the preferred linker can be constructed by connectingpreferred structures shown for each part of the linker explained above.As for the linker structure, those with the following structure can bepreferably used. Meanwhile, the left terminal of these structures is theconnecting position with the antibody and the right terminal is theconnecting position with the drug.

-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.

Among these, more preferred are the following.

-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.

Particularly preferred include the following.

-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.

[Production Method]

Next, explanations are given for the representative method for producingthe antibody-drug conjugate of the present invention or a productionintermediate thereof. Meanwhile, the compounds are hereinbelow describedwith the compound number shown in each reaction formula. Specifically,they are referred to as a “compound of the formula (1)”, a “compound(1)”, or the like. The compounds with numbers other than those are alsodescribed similarly.

1. Production Method 1

The antibody-drug conjugate represented by the formula (1) in which theantibody is connected to the drug-linker structure via thioether can beproduced by the following method, for example.

[In the formula, AB represents an antibody having a sulfhydryl group,and L¹′ represents L¹ linker structure in which the linker terminal is amaleimidyl group (formula shown below)

(in the formula, the nitrogen atom is the connecting position), andspecifically represents a group in which the -(Succinimid-3-yl-N)—moiety in -(Succinimid-3-yl-N)—(CH₂)n³—C(═O)— of L¹ is a maleimidylgroup. Further, the —(NH-DX) represents a structure represented by thefollowing formula:

and it represents a group that is derived by removing one hydrogen atomof the amino group at position 1 of exatecan.]

Further, the compound of the formula (1) in the above reaction formulamay be interpreted as a structure in which one structure moietycorresponding from the drug to the linker terminal connects to oneantibody. However, it is only a description given for the sake ofconvenience, and there are actually many cases in which a plurality ofthe structure moieties are connected to one antibody molecule. The sameapplies to the explanation of the production method described below.

The antibody-drug conjugate (1) can be produced by reacting the compound(2), which is obtainable by the method described below, with theantibody (3a) having a sulfhydryl group.

The antibody (3a) having a sulfhydryl group can be obtained by a methodwell known in the art (Hermanson, G. T, Bioconjugate Techniques, pp.56-136, pp. 456-493, Academic Press (1996)). Examples include: Traut'sreagent is reacted with the amino group of the antibody; N-succinimidylS-acetylthioalkanoates are reacted with the amino group of the antibodyfollowed by reaction with hydroxylamine; after reacting withN-succinimidyl 3-(pyridyldithio)propionate, the antibody is reacted witha reducing agent; the antibody is reacted with a reducing agent such asdithiothreitol, 2-mercaptoethanol, and tris(2-carboxyethyl)phosphinehydrochloride (TCEP) to reduce the disulfide bond in the hinge part inthe antibody to form a sulfhydryl group, but it is not limited thereto.

Specifically, using 0.3 to 3 molar equivalents of TCEP as a reducingagent per disulfide at the hinge part in the antibody and reacting withthe antibody in a buffer solution containing a chelating agent, theantibody with partially or completely reduced disulfide at the hingepart in the antibody can be obtained. Examples of the chelating agentinclude ethylenediamine tetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). It can be used at the concentration of 1 mM to20 mM. Examples of the buffer solution which may be used include asolution of sodium phosphate, sodium borate, or sodium acetate.Specifically, by reacting the antibody with TCEP at 4° C. to 37° C. for1 to 4 hours, the antibody (3a) having a partially or completely reducedsulfhydryl group can be obtained.

Meanwhile, by conducting the reaction for adding a sulfhydryl group to adrug-linker moiety, the drug-linker moiety can be conjugated by athioether bond.

Using 2 to 20 molar equivalents of the compound (2) per the antibody(3a) having a sulfhydryl group, the antibody-drug conjugate (1) in which2 to 8 drug molecules are conjugated per antibody molecule can beproduced. Specifically, it is sufficient that the solution containingthe compound (2) dissolved therein is added to a buffer solutioncontaining the antibody (3a) having a sulfhydryl group for the reaction.Herein, examples of the buffer solution which may be used include sodiumacetate solution, sodium phosphate, and sodium borate. The pH for thereaction is 5 to 9, and more preferably the reaction is performed nearpH 7. Examples of the solvent for dissolving the compound (2) include anorganic solvent such as dimethyl sulfoxide (DMSO), dimethylformamide(DMF), dimethyl acetamide (DMA), and N-methyl-2-pyridone (NMP).

It is sufficient that the organic solvent solution containing thecompound (2) dissolved therein is added at 1 to 20% v/v to a buffersolution containing the antibody (3a) having a sulfhydryl group for thereaction. The reaction temperature is 0 to 37° C., more preferably 10 to25° C., and the reaction time is 0.5 to 2 hours. The reaction can beterminated by deactivating the reactivity of unreacted compound (2) witha thiol-containing reagent. Examples of the thiol-containing reagentinclude cysteine and N-acetyl-L-cysteine (NAC). More specifically, 1 to2 molar equivalents of NAC are added to the compound (2) used and, byincubating at room temperature for 10 to 30 minutes, the reaction can beterminated.

The produced antibody-drug conjugate (1) can, after concentration,buffer exchange, purification, and measurement of antibody concentrationand average number of conjugated drug molecules per antibody moleculeaccording to common procedures described below, be subjected toidentification of the antibody-drug conjugate (1).

Common Procedure A: Concentration of Aqueous Solution of Antibody orAntibody-Drug Conjugate

To a Amicon Ultra (50,000 MWCO, Millipore Co.) container, a solution ofantibody or antibody-drug conjugate was added and the solution of theantibody or antibody-drug conjugate was concentrated by centrifugation(centrifuge for 5 to 20 minutes at 2000 G to 3800 G) using a centrifuge(Allegra X-15R, Beckman Coulter, Inc.).

Common Procedure B: Measurement of Antibody Concentration

Using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.),measurement of the antibody concentration was performed according to themethod defined by the manufacturer. At that time, a 280 nm absorptioncoefficient different for each antibody was used (1.3 mLmg⁻¹ cm⁻¹ to 1.8mLmg⁻¹ cm⁻¹)

Common Procedure C-1: Buffer Exchange for Antibody

NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation)using Sephadex G-25 carrier was equilibrated with phosphate buffer (10mM, pH 6.0; it is referred to as PBS6.0/EDTA in the specification)containing sodium chloride (137 mM) and ethylene diamine tetraaceticacid (EDTA, 5 mM) according to the method defined by the manufacturer.Aqueous solution of the antibody was applied in an amount of 2.5 mL tosingle NAP-25 column, and then the fraction (3.5 mL) eluted with 3.5 mLof PBS6.0/EDTA was collected. The resulting fraction was concentrated bythe Common procedure A. After measuring the concentration of theantibody using the Common procedure B, the antibody concentration wasadjusted to 10 mg/mL using PBS6.0/EDTA.

Common Procedure C-2: Buffer Exchange for Antibody

NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation)using Sephadex G-25 carrier was equilibrated with phosphate buffer (50mM, pH 6.5; it is referred to as PBS6.5/EDTA in the specification)containing sodium chloride (50 mM) and EDTA (2 mM) according to themethod defined by the manufacturer. Aqueous solution of the antibody wasapplied in an amount of 2.5 mL to single NAP-25 column, and then thefraction (3.5 mL) eluted with 3.5 mL of PBS6.5/EDTA was collected. Theresulting fraction was concentrated by the Common procedure A. Aftermeasuring the concentration of the antibody using the Common procedureB, the antibody concentration was adjusted to 20 mg/mL usingPBS6.5/EDTA.

Common Procedure D: Purification of Antibody-Drug Conjugate

NAP-25 column was equilibrated with any buffer selected fromcommercially available phosphate buffer (PBS7.4, Cat. No. 10010-023,Invitrogen), sodium phosphate buffer (10 mM, pH 6.0; it is referred toas PBS6.0) containing sodium chloride (137 mM), and acetate buffercontaining sorbitol (5%) (10 mM, pH 5.5; it is referred to as ABS in thespecification). Aqueous solution of the antibody-drug conjugate reactionwas applied in an amount of about 1.5 mL to the NAP-25 column, and theneluted with the buffer in an amount defined by the manufacturer tocollect the antibody fraction. The collected fraction was again appliedto the NAP-25 column and, by repeating 2 to 3 times in total the gelfiltration purification process for eluting with buffer, theantibody-drug conjugate excluding non-conjugated drug linker and alow-molecular-weight compound (tris(2-carboxyethyl)phosphinehydrochloride (TCEP), N-acetyl-L-cysteine (NAC), and dimethyl sulfoxide)was obtained.

Common Procedure E: Measurement of Antibody Concentration inAntibody-Drug Conjugate and Average Number of Conjugated Drug MoleculesPer Antibody Molecule (1).

The conjugated drug concentration in the antibody-drug conjugate can becalculated by measuring UV absorbance of an aqueous solution of theantibody-drug conjugate at two wavelengths of 280 nm and 370 nm,followed by performing the calculation shown below.

Because the total absorbance at any wavelength is equal to the sum ofthe absorbance of every light-absorbing chemical species that arepresent in the system (additivity of absorbance), when the molarabsorption coefficients of the antibody and the drug remain the samebefore and after conjugation between the antibody and the drug, theantibody concentration and the drug concentration in the antibody-drugconjugate are expressed by the following equations.

A ₂₈₀ =A _(D,280) +A _(A,280)=ε_(D,280) C _(D)+ε_(A,280) C_(A)  Equation (I)

A ₃₇₀ =A _(D,370) +A _(A,370)=ε_(D,370) C _(D)+ε_(A,370) C_(A)  Equation (II)

In the above, A₂₈₀ represents the absorbance of an aqueous solution ofthe antibody-drug conjugate at 280 nm, A₃₇₀ represents the absorbance ofan aqueous solution of the antibody-drug conjugate at 370 nm, A_(A,280)represents the absorbance of an antibody at 280 nm, A_(A,370) representsthe absorbance of an antibody at 370 nm, A_(D,280) represents theabsorbance of a conjugate precursor at 280 nm, A_(D,370) represents theabsorbance of a conjugate precursor at 370 nm, ε_(A,280) represents themolar absorption coefficient of an antibody at 280 nm, ε_(A,370)represents the molar absorption coefficient of an antibody at 370 nm,ε_(D,280) represents the molar absorption coefficient of a conjugateprecursor at 280 nm, ε_(D,370) represents the molar absorptioncoefficient of a conjugate precursor at 370 nm, C_(A) represents theantibody concentration in an antibody-drug conjugate, and C_(D)represent the drug concentration in an antibody-drug conjugate.

As for ε_(A,280), ε_(A,370), ε_(D,280), and ε_(D,370) in the above,previously prepared values (estimated values based on calculation ormeasurement values obtained by UV measurement of the compounds) areused. For example, ε_(A,280) can be estimated from the amino acidsequence of an antibody using a known calculation method (ProteinScience, 1995, vol. 4, 2411-2423). ε_(A,370) is generally zero. InExamples, as for the molar absorption coefficient of trastuzumab,ε_(A,280)=215400 (estimated value based on calculation) and ε_(A,370)=0were used. ε_(D,280) and ε_(D,370) can be obtained based onLambert-Beer's law (Absorbance=molar concentration×molar absorptioncoefficient×cell path length) by measuring the absorbance of a solutionin which the conjugate precursor to be used is dissolved at a certainmolar concentration. As for the molar absorption coefficient of a druglinker in the Examples, ε_(D,280)=5000 (measured average value) andε_(D,370)=19000 (measured average value) were used, unless otherwisespecified. By measuring A₂₈₀ and A₃₇₀ of an aqueous solution of theantibody-drug conjugate and solving the simultaneous equations (I) and(II) using the values, C_(A) and C_(D) can be obtained. Further, bydividing C_(D) by C_(A), the average number of conjugated drug moleculesper antibody molecule can be obtained.

Common Procedure F: Measurement (2) of Average Number of Conjugated DrugMolecules Per Antibody Molecule in Antibody-Drug Conjugate.

The average number of conjugated drug molecules per antibody molecule inthe antibody-drug conjugate can also be determined by high-performanceliquid chromatography (HPLC) analysis using the following method inaddition to the aforementioned Common procedure E.

[F-1. Preparation of Sample for HPLC Analysis (Reduction ofAntibody-Drug Conjugate)]

An antibody-drug conjugate solution (about 1 mg/mL, 60 μL) is mixed withan aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). A sample inwhich the disulfide bond between the L chain and the H chain of theantibody-drug conjugate has been cleaved by incubating the mixture for30 minutes at 37° C. is used in HPLC analysis.

[F-2. HPLC Analysis]

The HPLC analysis is performed under the following measurementconditions:

HPLC system: Agilent 1290 HPLC system (Agilent Technologies, Inc.)

Detector: ultraviolet absorption spectrometer (measurement wavelength:280 nm)

Column: PLRP-S(2.1×50 mm, 8 m, 1000 angstroms; Agilent Technologies,Inc., P/N PL1912-1802)

Column temperature: 80° C.

Mobile phase A: aqueous solution containing 0.04% trifluoroacetic acid(TFA)

Mobile phase B: acetonitrile solution containing 0.04% TFA

Gradient program: 29%-36% (0-12.5 min), 36%-42% (12.5-15 min), 42%-29%(15-15.1 min), and 29%-29% (15.1-25 min)

Sample injection volume: 15 μL

[F-3. Data Analysis]

[F-3-1] Compared with non-conjugated antibody L (L₀) and H (H₀) chains,drug-conjugated L (L chain connected to one drug molecule: L₁) and H (Hchain connected to one drug molecule: H₁, H chain connected to two drugmolecule: H₂, H chain connected to three drug molecules: H₃) chainsexhibit higher hydrophobicity in proportion to the number of conjugateddrug molecules and thus have a larger retention time. These chains aretherefore eluted in the order of L₀ and L₁ or H₀, H₁, H₂, and H₃.Detection peaks can be assigned to any of L₀, L₁, H₀, H₁, H₂, and H₃ bythe comparison of retention times with L₀ and H₀.[F-3-2] Since the drug linker has UV absorption, peak area values arecorrected in response to the number of conjugated drug linker moleculesaccording to the following expression using the molar absorptioncoefficients of the L or H chain and the drug linker.

$\begin{matrix}{\begin{matrix}{{Corrected}\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {the}} \\{{peak}\mspace{14mu} {area}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} L\mspace{14mu} {chain}\mspace{14mu} ({Li})}\end{matrix} = {{Peak}\mspace{14mu} {area} \times \frac{{Molar}\mspace{14mu} {absorption}\mspace{14mu} {coefficient}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} L\mspace{14mu} {chain}}{\begin{matrix}{{{Molar}\mspace{14mu} {absorption}\mspace{14mu} {coefficient}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} L\mspace{14mu} {chain}} +} \\{{The}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {conjugated}\mspace{14mu} {drug}\mspace{14mu} {molecules} \times} \\{{Molar}\mspace{14mu} {absorption}\mspace{14mu} {coefficient}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {drug}\mspace{14mu} {linker}}\end{matrix}}}} & \left\lbrack {{Expression}\mspace{14mu} 1} \right\rbrack \\{\begin{matrix}{{Corrected}\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {the}} \\{{peak}\mspace{14mu} {area}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} H\mspace{14mu} {chain}\mspace{14mu} ({Hi})}\end{matrix} = {{Peak}\mspace{14mu} {area} \times \frac{{Molar}\mspace{14mu} {absorption}\mspace{14mu} {coefficient}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} H\mspace{14mu} {chain}}{\begin{matrix}{{{Molar}\mspace{14mu} {absorption}\mspace{14mu} {coefficient}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} H\mspace{14mu} {chain}} +} \\{{The}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {conjugated}\mspace{14mu} {drug}\mspace{14mu} {molecules} \times} \\{{Molar}\mspace{14mu} {absorption}\mspace{14mu} {coefficient}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {drug}\mspace{14mu} {linker}}\end{matrix}}}} & \left\lbrack {{Expression}\mspace{14mu} 2} \right\rbrack\end{matrix}$

Here, as for the molar absorption coefficient (280 nm) of the L or Hchain of each antibody, a value estimated from the amino acid sequenceof the L or H chain of each antibody by a known calculation method(Protein Science, 1995, vol. 4, 2411-2423) can be used. In the case oftrastuzumab, a molar absorption coefficient of 26150 and a molarabsorption coefficient of 81290 were used as estimated values for the Land H chains, respectively, according to its amino acid sequence. As forthe molar absorption coefficient (280 nm) of the drug linker, themeasured molar absorption coefficient (280 nm) of a compound in whichthe maleimide group was converted to succinimide thioether by thereaction of each drug linker with mercaptoethanol or N-acetylcysteinewas used.

[F-3-3] The peak area ratio (%) of each chain is calculated for thetotal of the corrected values of peak areas according to the followingexpression.

$\begin{matrix}{{{{{{Peak}\mspace{14mu} {area}\mspace{14mu} {ratio}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} L\mspace{14mu} {chain}} = {\frac{A_{Li}}{A_{L\; 0} + A_{L\; 1}} \times 100}}{Peak}\mspace{14mu} {area}\mspace{14mu} {ratio}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} H\mspace{14mu} {chain}} = {\frac{A_{Hi}}{A_{H\; 0} + A_{H\; 1} + A_{H\; 2} + A_{H\; 3}} \times 100}}{{{Corrected}\mspace{14mu} {values}\mspace{14mu} {of}\mspace{14mu} {respective}\mspace{14mu} {peak}\mspace{14mu} {areas}\mspace{14mu} {of}\mspace{14mu} A_{Li}},{A_{Hi}\text{:}L_{i}},H_{i}}} & \left\lbrack {{Expression}\mspace{14mu} 3} \right\rbrack\end{matrix}$

[F-3-4] The average number of conjugated drug molecules per antibodymolecule in the antibody-drug conjugate is calculated according to thefollowing expression.

Average number of conjugated drug molecules=(L₀ peak area ratio×0+L₀peak area ratio×1+H₀ peak area ratio×0+H₁ peak area ratio×1+H₂ peak arearatio×2+H₃ peak area ratio×3)/100×2

The production intermediate compound used in Production method 1 isdescribed below. The compound represented by the formula (2) inProduction method 1 is a compound represented by the following formula:

(maleimid-N-yl)-(CH₂)n ³—C(═O)-L²-L^(P)-NH—(CH₂)n ¹-L^(a)-(CH₂)n²—C(═O)—(NH-DX).

In the formula,

n³ represents an integer of 2 to 8,L² represents —NH—(CH₂CH₂—O) n⁴—CH₂CH₂—C(═O)— or a single bond,

wherein n⁴ represents an integer of 1 to 6,

L^(P) represents a peptide residue consisting of 2 to 7 amino acidsselected from phenylalanine, glycine, valine, lysine, citrulline,serine, glutamic acid, and aspartic acid,n¹ represents an integer of 0 to 6,n² represents an integer of 0 to 5,L^(a) represents —O— or a single bond,(maleimid-N-yl)- is a maleimidyl group(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl group) represented by thefollowing formula:

wherein the nitrogen atom is the connecting position, and—(NH-DX) is a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is theconnecting position.

When L² is a single bond or —NH—(CH₂CH₂—O) n⁴—CH₂CH₂—C(═O)—, it ispreferred as a production intermediate that n⁴ should be an integer of 2to 4.

As for the peptide residue of L^(P), a compound having a peptide residueconsisting of an amino acid selected from phenylalanine, glycine,valine, lysine, citrulline, serine, glutamic acid, and aspartic acid ispreferred as a production intermediate. Among those peptide residues, acompound in which L^(P) is a peptide residue consisting of 4 amino acidsis preferred as a production intermediate. More specifically, a compoundin which L^(P) is the tetrapeptide residue -GGFG- is preferred as aproduction intermediate.

Further, as for the —NH—(CH₂)n¹-L^(a)-(CH₂)n², a compound having—NH—CH₂CH₂—, —NH—CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂CH₂—,—NH—CH₂—O—CH₂—, or —NH—CH₂CH₂—O—CH₂— is preferred as a productionintermediate. A compound having —NH—CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—, or—NH—CH₂CH₂—O—CH₂ is more preferred.

A compound represented by the formula (2) in which n³ is an integer of 2to 5, L² is a single bond, and —NH—(CH₂)n¹-L^(a)-(CH₂)n²- is—NH—CH₂CH₂—, —NH—CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂CH₂—,—NH—CH₂—O—CH₂—, or —NH—CH₂CH₂—O—CH₂— is preferred as a productionintermediate. A compound in which —NH—(CH₂)n¹-L^(a)-(CH₂)n²- is—NH—CH₂CH₂—, —NH—CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—, or —NH—CH₂CH₂—O—CH₂— ismore preferred. A compound in which n³ is an integer of 2 or 5 isfurther preferred.

A compound represented by the formula (2) in which n³ is an integer of 2to 5, L² is —NH—(CH₂CH₂—O)n⁴—CH₂CH₂—C(═O)—, n⁴ is an integer of 2 to 4,and —NH—(CH₂)n¹-L^(a)-(CH₂)n²- is —NH—CH₂CH₂—, —NH—CH₂CH₂CH₂—,—NH—CH₂CH₂CH₂CH₂—, —NH—CH₂CH₂CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—, or—NH—CH₂CH₂—O—CH₂— is preferred as a production intermediate. A compoundin which n⁴ is an integer of 2 or 4 is more preferred. A compound inwhich —NH—(CH₂)n¹-L^(a)-(CH₂)n²- is —NH—CH₂CH₂CH₂—, —NH—CH₂—O—CH₂—, or—NH—CH₂CH₂—O—CH₂— is further preferred.

Preferred examples of an intermediate useful in producing such acompound of the present invention can include the following.

(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)

The anti-HER2 antibody-drug conjugate of the present invention can beproduced by reacting a drug-linker compound selected from theabove-mentioned production intermediate compound group with an anti-HER2antibody or a reactive derivative thereof to thereby form a thioetherbond at a disulfide bond site present in the hinge part of the anti-HER2antibody. In this case, the reactive derivative of the anti-HER2antibody is preferably used, and a reactive derivative obtained byreducing the anti-HER2 antibody is particularly preferred.

The following are compounds more preferred as a production intermediate.

(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂—CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX)

Among the above-mentioned intermediate compound group, a compoundrepresented by the following formula:

(maleimid-N-yl)-CH₂CH₂—C(═O)—NH—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—(NH-DX),(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX),or(maleimid-N-yl)-CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—(NH-DX),is a further preferred compound.

In order to secure the amount of the conjugate, a plurality ofconjugates obtained under similar production conditions to have anequivalent number of drugs (e.g., about ±1) can be mixed to prepare newlots. In this case, the average number of drugs falls between theaverage numbers of drugs in the conjugates before the mixing.

2. Production Method 2

The compound represented by the formula (2) as an intermediate used inthe previous production method and a pharmacologically acceptable saltthereof can be produced by the following method, for example.

In the formula, L¹′ represents a terminal maleimidyl group, and P¹, P²,and P³ each represent a protecting group.

The compound (6) can be produced by derivatizing the carboxylic acid (5)into an active ester, mixed acid anhydride, acid halide, or the like andreacting it with NH₂-DX(4) or a pharmacologically acceptable saltthereof. NH₂-DX (4) indicates exatecan (chemical name:(1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-10,13(9H,15H)-dione).

Reaction reagents and conditions that are commonly used for peptidesynthesis can be employed for the reaction. There are various kinds ofactive ester. For example, it can be produced by reacting phenols suchas p-nitrophenol, N-hydroxy benzotriazole, N-hydroxy succinimide, or thelike, with the carboxylic acid (5) using a condensing agent such asN,N′-dicyclohexylcarbodiimide or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Further,the active ester can be also produced by a reaction of the carboxylicacid (5) with pentafluorophenyl trifluoroacetate or the like; a reactionof the carboxylic acid (5) with 1-benzotriazolyloxytripyrrolidinophosphonium hexafluorophosphite; a reaction of thecarboxylic acid (5) with diethyl cyanophosphonate (salting-in method); areaction of the carboxylic acid (5) with triphenylphosphine and2,2′-dipyridyl disulfide (Mukaiyama's method); a reaction of thecarboxylic acid (5) with a triazine derivative such as4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM); or the like. Further, the reaction can be also performed by,e.g., an acid halide method by which the carboxylic acid (5) is treatedwith an acid halide such as thionyl chloride and oxalyl chloride in thepresence of a base.

By reacting the active ester, mixed acid anhydride, or acid halide ofthe carboxylic acid (5) obtained as above with the compound (4) in thepresence of a suitable base in an inert solvent at −78° C. to 150° C.,the compound (6) can be produced. (Meanwhile, “inert solvent” indicatesa solvent which does not inhibit a reaction for which the solvent isused.)

Specific examples of the base used for each step described above includea carbonate, alkoxide, hydroxide or hydride of an alkali metal or analkali earth metal such as sodium carbonate, potassium carbonate, sodiumethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide,sodium hydride, and potassium hydride; an organometallic baserepresented by an alkyl lithium such as n-butyl lithium, or dialkylaminolithium such as lithium diisopropylamide; an organometallic base ofbissilylamine such as lithium bis(trimethylsilyl)amide; and an organicbase including a tertiary amine or a nitrogen-containing heterocycliccompound such as pyridine, 2,6-lutidine, collidine,4-dimethylaminopyridine, triethylamine, N-methyl morpholine,diisopropylethylamine, and diazabicyclo[5.4.0]undec-7-ene (DBU).

Examples of the inert solvent which is used for the reaction of thepresent invention include a halogenated hydrocarbon solvent such asdichloromethane, chloroform, and carbon tetrachloride; an ether solventsuch as tetrahydrofuran, 1,2-dimethoxyethane, and dioxane; an aromatichydrocarbon solvent such as benzene and toluene; and an amide solventsuch as N,N-dimethylformamide, N,N-dimethylacetamide, andN-methylpyrrolidin-2-one. In addition to these, a sulfoxide solvent suchas dimethyl sulfoxide and sulfolane; and a ketone solvent such asacetone and methyl ethyl ketone and an alcohol solvent such as methanoland ethanol may be used in some cases. Further, a mixed solvent thereofcan also be used.

As for the protecting group P¹ for the terminal amino group of thecompound (6), a protecting group for an amino group which is generallyused for peptide synthesis, for example, a tert-butyloxy carbonyl group,a 9-fluorenylmethyloxy carbonyl group, or a benzyloxy carbonyl group,can be used. Examples of other protecting groups for an amino groupinclude an alkanoyl group such as an acetyl group; an alkoxycarbonylgroup such as a methoxycarbonyl group and an ethoxycarbonyl group; anarylmethoxy carbonyl group such as a paramethoxybenzyloxy carbonylgroup, and a para (or ortho)nitrobenzyloxy carbonyl group; an arylmethylgroup such as a benzyl group and a triphenyl methyl group; an aroylgroup such as a benzoyl group; and an aryl sulfonyl group such as a2,4-dinitrobenzene sulfonyl group and a orthonitrobenzene sulfonylgroup. The protecting group P¹ can be selected depending on, e.g., theproperties of the compound having the amino group to be protected.

By deprotecting the protecting group P¹ for the terminal amino group ofthe compound (6) obtained, the compound (7) can be produced. In thedeprotection, reagents and conditions can be selected depending on theprotecting group.

The compound (9) can be produced by derivatizing the peptide carboxylicacid (8) having the N terminal protected with P² into an active ester,mixed acid anhydride, or the like and reacting it with the compound (7)obtained. The reaction conditions, reagents, base, and inert solventused for forming a peptide bond between the peptide carboxylic acid (8)and the compound (7) can be suitably selected from those described forthe synthesis of the compound (6). The protecting group P² can besuitably selected from those described for the protecting group of thecompound (6), and the selection can be made based on, e.g., theproperties of the compound having the amino group to be protected. As itis generally used for peptide synthesis, by repeating sequentially thereaction and deprotection of the amino acid or peptide constituting thepeptide carboxylic acid (8) for elongation, the compound (9) can be alsoproduced.

By deprotecting the protecting group P² for the amino group of thecompound (9) obtained, the compound (10) can be produced. In thedeprotection, reagents and conditions can be selected depending on theprotecting group.

It is possible to produce the compound (2) by derivatizing thecarboxylic acid (11) into an active ester, mixed acid anhydride, acidhalide, or the like and reacting it with the compound (10) obtained. Thereaction conditions, reagents, base, and inert solvent used for forminga peptide bond between the carboxylic acid (11) and the compound (10)can be suitably selected from those described for the synthesis of thecompound (6).

The compound (9) can be also produced by the following method, forexample.

The compound (13) can be produced by derivatizing the peptide carboxylicacid (8) having the N terminal protected with P² into an active ester,mixed acid anhydride, or the like and reacting it in the presence of abase with the amine compound (12) having the carboxy group protectedwith P³. The reaction conditions, reagents, base, and inert solvent usedfor forming a peptide bond between the peptide carboxylic acid (8) andthe compound (12) can be suitably selected from those described for thesynthesis of the compound (6). The protecting group P² for the aminogroup of the compound (13) is not particularly limited as long as it isa protecting group generally used.

Specifically, examples of the protecting group for a hydroxyl group caninclude an alkoxymethyl group such as a methoxymethyl group; anarylmethyl group such as a benzyl group, a 4-methoxybenzyl group, and atriphenylmethyl group; an alkanoyl group such as an acetyl group; anaroyl group such as a benzoyl group; and a silyl group such as atert-butyl diphenylsilyl group. Carboxy group can be protected, e.g., asan ester with an alkyl group such as a methyl group, an ethyl group, anda tert-butyl group, an allyl group, or an arylmethyl group such as abenzyl group. Examples of the protecting group for an amino group caninclude: an alkyloxy carbonyl group such as a tert-butyloxy carbonylgroup, a methoxycarbonyl group, and an ethoxycarbonyl group; anallyloxycarbonyl group, or an arylmethoxy carbonyl group such as a9-fluorenylmethyloxy carbonyl group, a benzyloxy carbonyl group, aparamethoxybenzyloxy carbonyl group, and a para (or ortho)nitrobenzyloxycarbonyl group; an alkanoyl group such as an acetyl group; an arylmethylgroup such as a benzyl group and a triphenyl methyl group; an aroylgroup such as a benzoyl group; and an aryl sulfonyl group such as a2,4-dinitrobenzene sulfonyl group or an orthonitrobenzene sulfonylgroup.

As for the protecting group P³ for a carboxy group, a protecting groupcommonly used as a protecting group for a carboxy group in organicsynthetic chemistry, in particular, peptide synthesis can be used.Specifically, it can be suitably selected from the protecting groupsdescribed above, for example, esters with an alkyl group such as amethyl group, an ethyl group, or a tert-butyl, allyl esters, and benzylesters.

In such cases, the protecting group for an amino group and theprotecting group for a carboxy group can be those preferably removed bya different method or different conditions. For example, arepresentative example includes a combination in which P² is atert-butyloxy carbonyl group and P³ is a benzyl group. The protectinggroups can be selected from the aforementioned ones depending on, e.g.,the properties of the compounds having the amino group and the carboxygroup to be protected. For removal of the protecting groups, reagentsand conditions can be selected depending on the protecting group.

By deprotecting the protecting group P³ for the carboxy group of thecompound (13) obtained, the compound (14) can be produced. In thedeprotection, reagents and conditions are selected depending on theprotecting group.

The compound (9) can be produced by derivatizing the compound (14)obtained into an active ester, mixed acid anhydride, acid halide, or thelike and reacting with the compound (4) in the presence of a base. Forthe reaction, reaction reagents and conditions that are generally usedfor peptide synthesis can be also used, and the reaction conditions,reagents, base, and inert solvent used for the reaction can be suitablyselected from those described for the synthesis of the compound (6).

The compound (2) can be also produced by the following method, forexample.

By deprotecting the protecting group P² for the amino group of thecompound (13), the compound (15) can be produced. In the deprotection,reagents and conditions can be selected depending on the protectinggroup.

The compound (16) can be produced by derivatizing the carboxylic acidderivative (11) into an active ester, mixed acid anhydride, acid halide,or the like and reacting it in the presence of a base with the compound(15) obtained. The reaction conditions, reagents, base, and inertsolvent used for forming an amide bond between the peptide carboxylicacid (11) and the compound (15) can be suitably selected from thosedescribed for the synthesis of the compound (6).

By deprotecting the protecting group for the carboxy group of thecompound (16) obtained, the compound (17) can be produced. Thedeprotection can be carried out similarly to the deprotection at thecarboxy group for producing the compound (14).

The compound (2) can be produced by derivatizing the compound (17) intoan active ester, mixed acid anhydride, acid halide, or the like andreacting it with the compound (4) in the presence of a base. For thereaction, reaction reagents and conditions that are generally used forpeptide synthesis can be also used, and the reaction conditions,reagents, base, and inert solvent used for the reaction can be suitablyselected from those described for the synthesis of the compound (6).

3. Production Method 3

The compound represented by the formula (2) of an intermediate can bealso produced by the following method.

In the formula, L^(1′) corresponds to L¹ having a structure in which theterminal is converted to a maleimidyl group, and P⁴ represents aprotecting group.

The compound (19) can be produced by derivatizing the compound (11) intoan active ester, mixed acid anhydride, or the like and reacting it inthe presence of a base with the peptide carboxylic acid (18) having theC terminal protected with P⁴. The reaction conditions, reagents, base,and inert solvent used for forming a peptide bond between the peptidecarboxylic acid (18) and the compound (11) can be suitably selected fromthose described for the synthesis of the compound (6). The protectinggroup P⁴ for the carboxy group of the compound (18) can be suitablyselected from the protecting groups described above.

By deprotecting the protecting group for the carboxy group of thecompound (19) obtained, the compound (20) can be produced. Thedeprotection can be performed similar to the deprotection of the carboxygroup for producing the compound (14).

The compound (2) can be produced by derivatizing the compound (20)obtained into an active ester, mixed acid anhydride, or the like andreacting it with the compound (7). For the reaction, reaction reagentsand conditions that are generally used for peptide synthesis can be alsoused, and the reaction conditions, reagents, base, and inert solventused for the reaction can be suitably selected from those described forthe synthesis of the compound (6).

4. Production Method 4

Hereinbelow, the method for producing the compound (10b) having n¹=1,L^(a)=O in the production intermediate (10) described in Productionmethod 2 is described in detail. The compound represented by the formula(10b), a salt or a solvate thereof can be produced according to thefollowing method, for example.

In the formula, L^(P) is as defined above, L represents an acyl groupwhich is an alkanoyl group such as an acetyl group or an aroyl groupsuch as benzoyl group, or a hydrogen atom, X and Y each represent anoligopeptide consisting of 1 to 3 amino acids, P⁵ and P⁷ each representa protecting group for an amino group, and P⁶ represents a protectinggroup for a carboxy group.

A compound represented by the formula (21) can be produced by using orapplying the method described in Japanese Patent Laid-Open No.2002-60351 or the literature (J. Org. Chem., Vol. 51, page 3196, 1986),and by conducting removal of the protecting groups or modification ofthe functional groups, if necessary. Furthermore, it can be alsoobtained by treating an amino acid with a protected terminal amino groupor an acid amide of an oligopeptide with protected amino group with analdehyde or a ketone.

By reacting the compound (21) with the compound (22) having a hydroxylgroup at a temperature ranging from under temperature conditions ofcooling to room temperature in an inert solvent in the presence of anacid or a base, the compound (23) can be produced.

Here, examples of the acid which may be used include an inorganic acidsuch as hydrofluoric acid, hydrochloric acid, sulfuric acid, nitricacid, phosphoric acid, and boric acid; an organic acid such as aceticacid, citric acid, paratoluene sulfonic acid, and methanesulfonic acid;and a Lewis acid such as tetrafluoroborate, zinc chloride, tin chloride,aluminum chloride, and iron chloride. Among these, a sulfonic acid, inparticular, paratoluene sulfonic acid is preferable. As for the base,any one of the already mentioned bases can be suitably selected andused. Preferred examples thereof include an alkali metal alkoxide suchas potassium tert-butoxide, an alkali metal hydroxide such as sodiumhydroxide and potassium hydroxide; an alkali metal or alkaline earthmetal hydride such as sodium hydride and potassium hydride; anorganometallic base represented by dialkylamino lithium such as lithiumdiisopropylamide; and an organometallic base of bissilylamine such aslithium bis(trimethylsilyl)amide.

Examples of the solvent to be used for the reaction include an ethersolvent such as tetrahydrofuran and 1,4-dioxane; and an aromatichydrocarbon solvent such as benzene and toluene. Those solvents can beprepared as a mixture with water.

Further, the protecting group for an amino group as exemplified by P⁵ isnot particularly limited provided it is a group commonly used forprotection of an amino group. Representative examples include theprotecting groups for an amino group that are described in Productionmethod 2. However, in the present reaction, there may be cases in whichthe protecting group for an amino group as exemplified by P⁵ is cleavedoff. In such cases, a protecting group can be introduced again byappropriately performing a reaction with a suitable reagent forprotecting an amino group as may be required.

The compound (24) can be produced by removing the protecting group P⁶ ofthe compound (23). Herein, representative examples of the protectinggroup for a carboxy group as exemplified by P⁶ are described inProduction method 2, and it can be appropriately selected from these. Inthe compound (23), it is desirable in this case that the protectinggroup P⁵ for an amino group and protecting group P⁶ for a carboxy groupare the protecting groups that can be removed by a different method ordifferent conditions. For example, a representative example includes acombination in which P⁵ is a 9-fluorenylmethyloxy carbonyl group and P⁶is a benzyl group. The protecting groups can be selected depending on,e.g., the properties of a compound having the amino group and thecarboxy group to be protected. For removal of the protecting groups,reagents and conditions are selected depending on the protecting group.

The compound (26) can be produced by derivatizing the carboxylic acid(24) into an active ester, mixed acid anhydride, acid halide, or thelike and reacting it with the compound (4) or a pharmacologicallyacceptable salt thereof to produce the compound (25) followed byremoving the protecting group P⁵ of the compound (25) obtained. For thereaction between the compound (4) and the carboxylic acid (24) and thereaction for removing the protecting group P⁶, the same reagents andreaction conditions as those described for Production method 2 can beused.

The compound (10b) can be produced by reacting the compound (26) with anamino acid with a protected terminal amino group or the oligopeptide(27) with a protected amino group to produce the compound (9b) andremoving the protecting group P⁷ of the compound (9b) obtained. Theprotecting group for an amino group as represented by P⁷ is notparticularly limited provided it is generally used for protection of anamino group. Representative examples thereof include the protectinggroups for an amino group that are described in Production method 2. Forremoving the protecting group, reagents and conditions are selecteddepending on the protecting group. For the reaction between the compound(26) and the compound (27), reaction reagents and conditions that arecommonly used for peptide synthesis can be employed. The compound (10b)produced by the aforementioned method can be derivatized into thecompound (1) of the present invention according to the method describedabove.

5. Production Method 5

Hereinbelow, the method for producing the compound (2) having n¹=1,n²=i, L^(a)=O in the production intermediate (2) described in Productionmethod 2 is described in detail. The compound represented by the formula(2), a salt or a solvate thereof can be produced according to thefollowing method, for example.

In the formula, L^(1′), L², L^(P) are as defined above, Z represents anoligopeptide consisting of 1 to 3 amino acids, P⁸ represents aprotecting group for an amino group, and P⁹ represents a protectinggroup for a carboxy group.

The compound (30) can be produced by removing the protecting group P⁸ ofthe amino acid or oligopeptide (28) with the protected terminal aminogroup and carboxy group to produce the compound (29) and reacting theobtained amine form (29) with the compound (11). The protecting groupfor an amino group as represented by P⁸ is not particularly limitedprovided it is a group commonly used for protection of an amino group.Representative examples include the protecting groups for an amino groupthat are described in Production method 2. Further, for removing theprotecting group P⁸, reagents and conditions can be selected dependingon the protecting group. For the reaction between the compound (29) andthe carboxylic acid (11), the same reagents and reaction conditions asthose described for Production method 2 can be used.

The production intermediate (2b) can be produced by removing theprotecting group P⁹ of the compound (30) to produce the compound (31)and reacting the obtained carboxylic acid (31) with the compound (26).The representative examples of the protecting group for a carboxy groupas represented by P⁸ are described in Production method 2. For thedeprotection reaction thereof, the same reagents and reaction conditionsas those described for Production method 2 can be used. For the reactionbetween the compound (26) and the carboxylic acid (31), reactionreagents and conditions that are generally used for peptide synthesiscan be also used. The compound (2b) produced by the aforementionedmethod can be derivatized into the compound (1) of the present inventionaccording to the method described above.

6. Production Method 6

Hereinbelow, a method for producing the compound (17b) having n¹=1,n²=i, L^(a)=O in the production intermediate (17) described inProduction method 2 is described in detail. The compound represented bythe formula (17b), a salt or a solvate thereof can be also producedaccording to the following method, for example.

In the formula, L^(1′), L², L^(P), X, Y, P⁵, P⁶, and P⁷ are as definedabove.

The compound (33) can be produced by deprotecting the protecting groupP⁵ for the amino group of the compound (23) having the protectedterminal amino group and carboxy group to produce the compound (32) andreacting the obtained amine derivative (32) with the oligopeptide (27)having a protected terminal amino group or a protected amino group. Theprotecting group for an amino group as represented by P⁵ is notparticularly limited provided it is a group commonly used for protectionof an amino group. Representative examples include the protecting groupsfor an amino group that are described in Production method 2. Further,for removing the protecting group P⁵, reagents and conditions can beselected depending on the protecting group. Herein, althoughrepresentative examples of the protecting group for a carboxy group asrepresented by P⁶ and the protecting group for an amino group asrepresented by P⁷ include the protecting groups for a carboxy group andan amino group that are described in Production method 2. It isdesirable that in the compound (33), the protecting group P⁶ for acarboxy group and the protecting group P⁷ for an amino group areprotecting groups that can be removed by the same method or the sameconditions. For example, a representative example includes a combinationin which P⁶ is a benzyl ester group and P⁷ is a benzyloxy carbonylgroup.

The compound (34) can be produced by removing the protecting group P⁶for the carboxy group of the compound (33) and the protecting group P⁷for the amino group of the compound (33). The compound (37) can be alsoproduced by sequentially removing the protecting group P⁶ for thecarboxy group and the protecting group P⁷ for the amino group, andfurthermore, the compound (34) can be produced simply by removing atonce both of the protecting groups P⁶ and P⁷ that can be removed by thesame method or the same conditions.

The compound (17b) can be produced by reacting the obtained compound(34) with the compound (11). For the reaction between the compound (34)and the compound (11), the same reagents and reaction conditions asthose described for Production method 2 can be used.

The anti-HER2 antibody-drug conjugate of the present invention, when itis left in air or recrystallized or purified, may absorb moisture orhave adsorption water or turn into a hydrate, and such compounds orsalts containing water are also included in the present invention.

Compounds labeled with various radioactive or non-radioactive isotopesare also included in the present invention. One or more atomsconstituting the antibody-drug conjugate of the present invention maycontain an atomic isotope at a non-natural ratio. Examples of atomicisotopes include deuterium (²H), tritium (³H), iodine-125 (¹²⁵I), andcarbon-14 (¹⁴C) Further, the compound of the present invention may beradioactive-labeled with a radioactive isotope such as tritium (³H),iodine-125 (¹²⁵I), carbon-14 (¹⁴C), copper-64 (⁶⁴Cu), zirconium-89(⁸⁹Zr), iodine-124 (¹²⁴I), fluorine-18 (¹⁸F), indium-111 (¹¹¹I),carbon-11 (¹¹C) and iodine-131 (¹³¹I). The compound labeled with aradioactive isotope is useful as a therapeutic or prophylactic agent, areagent for research such as an assay reagent and an agent for diagnosissuch as an in vivo diagnostic imaging agent. Without being related toradioactivity, any isotope variant type of the antibody-drug conjugateof the present invention is within the scope of the present invention.

[Drugs]

The anti-HER2 antibody-drug conjugate of the present invention exhibitscytotoxic activity against cancer cells, and thus, it can be used as adrug, particularly as a therapeutic agent and/or prophylactic agent forcancer.

That is, the anti-HER2 antibody-drug conjugate of the present inventioncan be selectively used as a drug for chemotherapy, which is a mainmethod for treating cancer, and as a result, can delay development ofcancer cells, inhibit growth thereof, and further kill cancer cells.This can allow cancer patients to be free from symptoms caused by canceror achieve improvement in QOL of cancer patients and attains atherapeutic effect by sustaining the lives of the cancer patients. Evenif the anti-HER2 antibody-drug conjugate of the present invention doesnot accomplish killing cancer cells, it can achieve higher QOL of cancerpatients while achieving longer-term survival, by inhibiting orcontrolling the growth of cancer cells.

In such drug therapy, it can be used as a drug alone and in addition, itcan be used as a drug in combination with an additional therapy inadjuvant therapy and can be combined with surgical operation,radiotherapy, hormone therapy, or the like. Furthermore, it can also beused as a drug for drug therapy in neoadjuvant therapy.

In addition to the therapeutic use as described above, an effect ofsuppressing the growth of small metastatic cancer cells and furtherkilling them can also be expected. Particularly, when the expression ofHER2 is confirmed in primary cancer cells, inhibition of cancermetastasis or a prophylactic effect can be expected by administering theanti-HER2 antibody-drug conjugate of the present invention. For example,an effect of inhibiting and killing cancer cells in a body fluid in thecourse of metastasis or an effect of, for example, inhibiting andkilling small cancer cells immediately after implantation in any tissuecan be expected. Accordingly, inhibition of cancer metastasis or aprophylactic effect can be expected, particularly, after surgicalremoval of cancer.

The anti-HER2 antibody-drug conjugate of the present invention can beexpected to exert a therapeutic effect by administration as systemictherapy to patients, and additionally, by local administration to cancertissues.

Examples of the cancer type to which the anti-HER2 antibody-drugconjugate of the present invention is applied can include lung cancer,urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer,pancreatic cancer, breast cancer, bladder cancer, gastric cancer,gastrointestinal stromal tumor, uterine cervix cancer, esophagealcancer, squamous cell carcinoma, peritoneal cancer, liver cancer,hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer,endometrial cancer, uterine cancer, salivary gland cancer, kidneycancer, vulval cancer, thyroid cancer, or penis cancer. The treatmentsubject of the anti-HER2 antibody-drug conjugate of the presentinvention is a cancer cell expressing, in a cancer cell as a treatmentsubject, HER2 protein which the antibody within the antibody-drugconjugate can recognize. The term “cancer expressing HER2 protein” asused in the present specification is a cancer containing cells havingHER2 protein on their cell surface. The HER2 protein is overexpressed invarious human tumors and can be evaluated using a method generallycarried out in the art, such as immunohistochemical staining method(IHC) for evaluating the overexpression of the HER2 protein, orfluorescence in situ hybridization method (FISH) for evaluatingamplification of the HER2 gene.

Further, the anti-HER2 antibody-drug conjugate of the present inventionexhibits an antitumor effect by recognizing, through its anti-HER2antibody, the HER2 protein expressed on the surface of cancer cells andinternalizing in the cancer cells. Thus, the treatment subject of theanti-HER2 antibody-drug conjugate of the present invention is notlimited to the “cancer expressing HER2 protein” and can also be, forexample, leukemia, malignant lymphoma, plasmacytoma, myeloma, orsarcoma.

The anti-HER2 antibody-drug conjugate of the present invention can bepreferably administered to a mammal, but it is more preferablyadministered to a human.

Substances used in a pharmaceutical composition containing anti-HER2antibody-drug conjugate of the present invention can be suitablyselected and applied from formulation additives or the like that aregenerally used in the art, in view of the dosage or administrationconcentration.

The anti-HER2 antibody-drug conjugate of the present invention can beadministered as a pharmaceutical composition containing at least onepharmaceutically suitable ingredient. For example, the pharmaceuticalcomposition above typically contains at least one pharmaceutical carrier(for example, sterilized liquid). Herein, the liquid includes, forexample, water and oil (petroleum oil and oil of animal origin, plantorigin, or synthetic origin. The oil may be, for example, peanut oil,soybean oil, mineral oil, or sesame oil. Water is a more typical carrierwhen the pharmaceutical composition above is intravenously administered.Saline solution, an aqueous dextrose solution, and an aqueous glycerolsolution can be also used as a liquid carrier, in particular, for aninjection solution. A suitable pharmaceutical vehicle can be selectedfrom ones known in the art. If desired, the composition above may alsocontain a trace amount of a moisturizing agent, an emulsifying agent, ora pH buffering agent. Examples of suitable pharmaceutical carrier aredisclosed in “Remington's Pharmaceutical Sciences” by E. W. Martin. Theformulations correspond to an administration mode.

Various delivery systems are known and they can be used foradministering the anti-HER2 antibody-drug conjugate of the presentinvention. Examples of the administration route can include intradermal,intramuscular, intraperitoneal, intravenous, and subcutaneous routes,but not limited thereto. The administration can be made by injection orbolus injection, for example. According to a specific preferredembodiment, the administration of the antibody-drug conjugate isperformed by injection. Parenteral administration is a preferredadministration route.

According to a representative embodiment, the pharmaceutical compositionis prescribed, as a pharmaceutical composition suitable for intravenousadministration to human, according to the conventional procedures. Thecomposition for intravenous administration is typically a solution in asterile and isotonic aqueous buffer solution. If necessary, the drug maycontain a solubilizing agent and local anesthetics to alleviate pain atinjection site (for example, lignocaine). Generally, the ingredientabove is provided individually as any one of lyophilized powder or ananhydrous concentrate contained in a container which is obtained bysealing in an ampoule or a sachet having an amount of the active agentor as a mixture in a unit dosage form. When the drug is to beadministered by injection, it may be administered from an injectionbottle containing water or saline of sterile pharmaceutical grade. Whenthe drug is administered by injection, an ampoule of sterile water orsaline for injection may be provided such that the aforementionedingredients are admixed with each other before administration.

The pharmaceutical composition of the present invention may be apharmaceutical composition containing only the anti-HER2 antibody-drugconjugate of the present invention or a pharmaceutical compositioncontaining the anti-HER2 antibody-drug conjugate and at least one cancertreating agent other than the conjugate. The anti-HER2 antibody-drugconjugate of the present invention can be administered with other cancertreating agents. The anti-cancer effect may be enhanced accordingly.Other anti-cancer agents used for such purpose may be administered to anindividual simultaneously with, separately from, or subsequently to theantibody-drug conjugate, and may be administered while varying theadministration interval for each. Examples of cancer treating agentsinclude 5-FU, pertuzumab, paclitaxel, carboplatin, cisplatin,gemcitabine, capecitabine, irinotecan (CPT-11), paclitaxel, docetaxel,pemetrexed, sorafenib, vinblastin, vinorelbine, everolims, tanespimycin,bevacizumab, oxaliplatin, lapatinib, ado-trastuzumab emtansine(T-DM1),or drugs described in International Publication No. WO 2003/038043,LH-RH analogues (leuprorelin, goserelin, or the like), estramustinephosphate, estrogen antagonists (tamoxifen, raloxifene, or the like),and aromatase inhibitors (anastrozole, letrozole, exemestane, or thelike), but are not limited as long as they are drugs having an antitumoractivity.

The pharmaceutical composition can be formulated into a lyophilizationformulation or a liquid formulation as a formulation having the desiredcomposition and required purity. When formulated as a lyophilizationformulation, it may be a formulation containing suitable formulationadditives that are used in the art. Also for a liquid formulation, itcan be formulated as a liquid formulation containing various formulationadditives that are used in the art.

The composition and concentration of the pharmaceutical composition mayvary depending on administration method. However, the anti-HER2antibody-drug conjugate contained in the pharmaceutical composition ofthe present invention can exhibit a pharmaceutical effect even at asmall dosage when the antibody-drug conjugate has a higher affinity foran antigen, that is, a higher affinity (=lower Kd value) in terms of thedissociation constant (that is, Kd value) for the antigen. Thus, fordetermining the dosage of the antibody-drug conjugate, the dosage can bedetermined in view of the situation relating to the affinity between theantibody-drug conjugate and antigen. When the antibody-drug conjugate ofthe present invention is administered to a human, for example, about0.001 to 100 mg/kg can be administered once or administered severaltimes with an interval of for 1 to 180 days.

at one time with an interval of 1 to 180 days

EXAMPLES

The present invention is specifically described in view of the examplesshown below. However, the present invention is not limited to these.Further, it is by no means interpreted in a limited way. Further, unlessspecifically described otherwise, the reagent, solvent, and startingmaterial described in the specification can be easily obtained from acommercial supplier.

Reference Example 1 Preparation of Trastuzumab

Fourteen vials of 440 mg/vial Herceptin (Genentech, Inc.) were dissolvedin 2 L of cation-exchange chromatography buffer A (25 mM citrate buffer,30 mM NaCl, pH 5.0) and filtered through a 0.2 m filter (MilliporeCorp.: Stericup 0.22 μm, GVPVDF Membrane). The samples were applied to acation-exchange chromatography column (SP Sepharose HP 240 ml, XK50column), followed by elution under a NaCl concentration linear gradientfrom 30 mM to 500 mM using cation-exchange chromatography buffer B (25mM citrate buffer, 500 mM NaCl, pH 5.0) to separate IgG monomerfractions. Monomer samples having a higher purity over 98% by sizeexclusion chromatography analysis were combined and concentrated withUF30K (Millipore Corp.: PELLICON XL Filter, BIOMAX 30K, PXB030A50), andthe buffer was replaced with CBS buffer (10 mM citrate/140 mM NaCl, pH6.0). The CBS buffer-replaced samples were filtered through a 0.2 mfilter (Sartorius AG: Minisart-Plus 0.2 μm, 17823K).

Reference Example 2 Production of Trastuzumab Emtansine T-DM1

SMCC derivatization of antibody: By using the Common procedure C-2(PBS6.5/EDTA was used as a buffer solution), Common procedure A, andCommon procedure B (as absorption coefficient at 280 nm, 1.37 mLmg⁻¹cm⁻¹ was used) described in Production method 1, replacement of bufferwith PBS6.5/EDTA was conducted on the trastuzumab produced in ReferenceExample 1 to prepare a solution containing trastuzumab (160.0 mg)dissolved in PBS6.5/EDTA (7.60 mL) in a 15 mL polypropylene tube.Subsequently, SMCC (1.84 mg) DMSO solution (0.40 mL; which correspondsto about 5.1 equivalents per antibody molecule) was added at roomtemperature. The reaction mixture was adjusted to have an antibodyconcentration of 20 mg/mL, and the reaction was carried out at roomtemperature by using a tube rotator (MTR-103, manufactured by AS ONECorporation) for 2 hours. This reaction solution was subjected topurification according to the Common procedure D-2 (PBS6.5/EDTA was usedas a buffer solution) to yield 12 mL of a solution containing 154.9 mgof the SMCC-derivatized antibody. Conjugation between antibody and druglinker: Adding PBS6.5/EDTA (2.56 mL) andN²-deacetyl-N²-(3-mercapto-1-oxopropyl)-maytansine (4.67 mg; DM1,Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14, p. 4392) DMA(dimethylacetamide) solution (0.93 mL; which corresponds to about 5.8equivalents per SMCC-derivatized antibody molecule) to the solutionobtained above in the 50 mL polypropylene tube at room temperature, thereaction solution was adjusted to an antibody concentration of 10 mg/mL,and the reaction was carried out at room temperature by using a tuberotator for 16.5 hours.

Purification procedure: The above solution was subjected to purificationusing the Common procedure D-1 using a sodium phosphate buffer solution(10 mM, pH 6.5) containing sodium chloride (137 mM) to yield 35 mL of asolution containing the target Reference Example compound.Physicochemical characterization: By using the Common procedure E usingUV absorbance at two wavelengths of 252 nm and 280 nm, the followingcharacteristic values were obtained.Antibody concentration: 4.14 mg/mL, antibody yield: 144.9 mg (91%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.0.

Example 1 Intermediate (1)

Process 1: tert-Butyl(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)carbamate

4-(tert-Butoxycarbonylamino)butanoic acid (0.237 g, 1.13 mmol) wasdissolved in dichloromethane (10 mL), N-hydroxysuccinimide (0.130 g,1.13 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (0.216 g, 1.13 mmol) were added and stirred for 1 hour.The reaction solution was added dropwise to an N,N-dimethylformamidesolution (10 mL) charged with methanesulfonic acid salt of exatecan(0.500 g, 0.94 mmol) and triethylamine (0.157 mL, 1.13 mmol), andstirred at room temperature for 1 day. The solvent was removed underreduced pressure and the residues obtained were purified by silica gelcolumn chromatography [chloroform-chloroform:methanol=8:2 (v/v)] toyield the titled compound (0.595 g, quantitative).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.2 Hz), 1.31 (9H, s), 1.58(1H, t, J=7.2 Hz), 1.66 (2H, t, J=7.2 Hz), 1.82-1.89 (2H, m), 2.12-2.21(3H, m), 2.39 (3H, s), 2.92 (2H, t, J=6.5 Hz), 3.17 (2H, s), 5.16 (1H,d, J=18.8 Hz), 5.24 (1H, d, J=18.8 Hz), 5.42 (2H, s), 5.59-5.55 (1H, m),6.53 (1H, s), 6.78 (1H, t, J=6.3 Hz), 7.30 (1H, s), 7.79 (1H, d, J=11.0Hz), 8.40 (1H, d, J=8.6 Hz).

MS (APCI) m/z: 621 (M+H)⁺

Process 2:4-Amino-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]butanamide

The compound (0.388 g, 0.61 mmol) obtained in Process 1 above wasdissolved in dichloromethane (9 mL). Trifluoroacetic acid (9 mL) wasadded and it was stirred for 4 hours. The solvent was removed underreduced pressure and the residues obtained were purified by silica gelcolumn chromatography [chloroform-partitioned organic layer ofchloroform:methanol:water=7:3:1 (v/v/v)] to yield trifluoroacetate ofthe titled compound (0.343 g, quantitative).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.2 Hz), 1.79-1.92 (4H, m),2.10-2.17 (2H, m), 2.27 (2H, t, J=7.0 Hz), 2.40 (3H, s), 2.80-2.86 (2H,m), 3.15-3.20 (2H, m), 5.15 (1H, d, J=18.8 Hz), 5.26 (1H, d, J=18.8 Hz),5.42 (2H, s), 5.54-5.61 (1H, m), 6.55 (1H, s), 7.32 (1H, s), 7.72 (3H,brs), 7.82 (1H, d, J=11.0 Hz), 8.54 (1H, d, J=8.6 Hz).

MS (APCI) m/z: 521 (M+H)+

Example 2 Antibody-Drug Conjugate (2)

Process 1:N-(tert-Butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

N-(tert-Butoxycarbonyl)glycylglycyl-L-phenylalanylglycine (0.081 g, 0.19mmol) was dissolved in dichloro methane (3 mL), N-hydroxysuccinimide(0.021 g, 0.19 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (0.036 g, 0.19 mmol) were added and then stirred for 3.5hours. The reaction solution was added dropwise to anN,N-dimethylformamide solution (1.5 mL) charged with the compound (0.080g, 0.15 mmol) obtained in Process 2 of Example 1, and stirred at roomtemperature for 4 hours. The solvent was removed under reduced pressureand the residues obtained were purified by silica gel columnchromatography [chloroform-chloroform:methanol=8:2 (v/v)] to yield thetitled compound (0.106 g, 73%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.36 (9H, s), 1.71(2H, m), 1.86 (2H, t, J=7.8 Hz), 2.15-2.19 (4H, m), 2.40 (3H, s), 2.77(1H, dd, J=12.7, 8.8 Hz), 3.02 (1H, dd, J=14.1, 4.7 Hz), 3.08-3.11 (2H,m), 3.16-3.19 (2H, m), 3.54 (2H, d, J=5.9 Hz), 3.57-3.77 (4H, m),4.46-4.48 (1H, m), 5.16 (1H, d, J=19.2 Hz), 5.25 (1H, d, J=18.8 Hz),5.42 (2H, s), 5.55-5.60 (1H, m), 6.53 (1H, s), 7.00 (1H, t, J=6.3 Hz),7.17-7.26 (5H, m), 7.31 (1H, s), 7.71 (1H, t, J=5.7 Hz), 7.80 (1H, d,J=11.0 Hz), 7.92 (1H, t, J=5.7 Hz), 8.15 (1H, d, J=8.2 Hz), 8.27 (1H, t,J=5.5 Hz), 8.46 (1H, d, J=8.2 Hz).

MS (APCI) m/z: 939 (M+H)⁺

Process 2:Glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (1.97 g, 2.10 mmol) obtained in Process 1 above wasdissolved in dichloromethane (7 mL). After adding trifluoroacetic acid(7 mL), it was stirred for 1 hour. The solvent was removed under reducedpressure, and the residues were charged with toluene for azeotropicdistillation. The residues obtained were purified by silica gel columnchromatography [chloroform-partitioned organic layer ofchloroform:methanol:water=7:3:1 (v/v/v)] to yield trifluoroacetate ofthe titled compound (1.97 g, 99%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.71-1.73 (2H, m),1.82-1.90 (2H, m), 2.12-2.20 (4H, m), 2.40 (3H, s), 2.75 (1H, dd,J=13.7, 9.4 Hz), 3.03-3.09 (3H, m), 3.18-3.19 (2H, m), 3.58-3.60 (2H,m), 3.64 (1H, d, J=5.9 Hz), 3.69 (1H, d, J=5.9 Hz), 3.72 (1H, d, J=5.5Hz), 3.87 (1H, dd, J=16.8, 5.9 Hz), 4.50-4.56 (1H, m), 5.16 (1H, d,J=19.2 Hz), 5.25 (1H, d, J=18.8 Hz), 5.42 (2H, s), 5.55-5.60 (1H, m),7.17-7.27 (5H, m), 7.32 (1H, s), 7.78-7.81 (2H, m), 7.95-7.97 (3H, m),8.33-8.35 (2H, m), 8.48-8.51 (2H, m).

MS (APCI) m/z: 839 (M+H)⁺

Process 3:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

To an N,N-dimethylformamide (1.2 mL) solution of the compound (337 mg,0.353 mmol) obtained in Process 2 above, triethylamine (44.3 mL, 0.318mmol) and N-succinimidyl 6-maleimide hexanoate (119.7 mg, 0.388 mmol)were added and stirred at room temperature for 1 hour. The solvent wasremoved under reduced pressure and the residues obtained were purifiedby silica gel column chromatography [chloroform-chloroform methanol=5:1(v/v)] to yield the titled compound as a pale yellow solid (278.0 mg,76%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.3 Hz), 1.12-1.22 (2H, m),1.40-1.51 (4H, m), 1.66-1.76 (2H, m), 1.80-1.91 (2H, m), 2.05-2.21 (6H,m), 2.39 (3H, s), 2.79 (1H, dd, J=14.0, 9.8 Hz), 2.98-3.21 (5H, m),3.55-3.77 (8H, m), 4.41-4.48 (1H, m), 5.15 (1H, d, J=18.9 Hz), 5.24 (1H,d, J=18.9 Hz), 5.40 (1H, d, J=17.1 Hz), 5.44 (1H, d, J=17.1 Hz),5.54-5.60 (1H, m), 6.53 (1H, s), 6.99 (2H, s), 7.20-7.27 (5H, m), 7.30(1H, s), 7.70 (1H, t, J=5.5 Hz), 7.80 (1H, d, J=11.0 Hz), 8.03 (1H, t,J=5.8 Hz), 8.08 (1H, t, J=5.5 Hz), 8.14 (1H, d, J=7.9 Hz), 8.25 (1H, t,J=6.1 Hz), 8.46 (1H, d, J=8.5 Hz).

MS (APCI) m/z: 1032 (M+H)⁺

Process 4: Antibody-Drug Conjugate (2)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure C-1 and Common procedure B (asabsorption coefficient at 280 nm, 1.37 mLmg⁻¹ cm⁻¹ was used) describedin Production method 1. The solution (3.0 mL) was placed in a 15 mLpolypropylene tube and charged with an aqueous solution of 10 mMtris(2-carboxyethyl)phosphine hydrochloride (TCEP, Tokyo ChemicalIndustry Co., Ltd.) (0.0934 mL; 4.6 equivalents per antibody molecule)and an aqueous solution of 1 M dipotassium hydrogen phosphate (NacalaiTesque, Inc.; 0.150 mL). After confirming that the solution had a pH of7.4±0.1, the disulfide bond at the hinge part in the antibody wasreduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating thesolution above at 22° C. for 10 minutes, a DMSO solution (0.187 mL; 9.2equivalents per antibody molecule) containing 10 mM of the compoundobtained in Process 3 was added thereto and incubated for conjugatingthe drug linker to the antibody at 22° C. for 40 minutes. Next, anaqueous solution (0.0374 mL; 18.4 equivalents per antibody molecule) ofN-acetylcysteine (NAC, Sigma-Aldrich Co. LLC) was added thereto andincubated at 22° C. to terminate the reaction of the drug linker foranother 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS6.0 was used as buffer solution) described inProduction method 1 to yield 6 mL of a solution containing the titledantibody-drug conjugate. After that, the solution was concentrated bythe Common procedure A.

Physicochemical characterization: By using the Common procedure Edescribed in Production method 1, the following characteristic valueswere obtained.

Antibody concentration: 3.21 mg/mL, antibody yield: 22.5 mg (75%), andaverage number of conjugated drug molecules (n) per antibody molecule:2.6.

Example 3 Antibody-Drug Conjugate (3)

Process 1: Antibody-Drug Conjugate (3)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.487mLmg⁻¹ cm⁻¹ was used). The solution (1.25 mL) was placed in a 1.5 mLpolypropylene tube and charged with an aqueous solution of 10 mM TCEP(0.039 mL; 4.6 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (0.0625 mL). Afterconfirming that the solution had a pH of 7.4±0.1, the disulfide bond atthe hinge part in the antibody was reduced by incubating at 37° C. for 1hour. Conjugation between antibody and drug linker: After adding DMSO(0.072 mL) and a DMSO solution containing 10 mM of the compound ofProcess 3 of Example 2 (0.078 mL; 9.2 equivalents per antibody molecule)to the above solution at room temperature, it was stirred by using atube rotator for conjugating the drug linker to the antibody at roomtemperature for 40 minutes. Next, an aqueous solution (0.0155 mL; 18.4equivalents per antibody molecule) of 100 mM NAC was added thereto andstirred at room temperature to terminate the reaction of the drug linkerfor another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D (ABS was used as buffer solution) described inProduction method 1 to yield 6 mL of a solution containing the compoundof interest. The solution was further concentrated by the Commonprocedure A. After that, by using the Common procedure E, the followingcharacteristic values were obtained.

Antibody concentration: 9.85 mg/mL, antibody yield: 6.9 mg (55%), andaverage number of conjugated drug molecules (n) per antibody molecule:7.3.

Example 4 Antibody-Drug Conjugate (4)

Process 1:N-[3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (80 mg, 0.084 mmol) of Example 1 was reacted in the samemanner as Process 3 of Example 2 by using N-succinimidyl 3-maleimidepropionate (24.6 mg, 0.0924 mmol) instead of N-succinimidyl 6-maleimidehexanoate to yield the titled compound as a pale yellow solid (60.0 mg,73%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.89 (3H, t, J=7.3 Hz), 1.70-1.78 (2H, m),1.81-1.94 (2H, m), 2.12-2.23 (4H, m), 2.42 (3H, s), 2.81 (1H, dd,J=13.7, 9.8 Hz), 3.01-3.15 (3H, m), 3.16-3.23 (2H, m), 3.30-3.35 (1H,m), 3.58-3.71 (6H, m), 3.71-3.79 (1H, m), 4.44-4.51 (1H, m), 5.19 (1H,d, J=19.0 Hz), 5.27 (1H, d, J=19.0 Hz), 5.43 (1H, d, J=17.6 Hz), 5.47(1H, d, J=17.6 Hz), 5.57-5.63 (1H, m), 6.56 (1H, s), 7.02 (2H, s),7.17-7.22 (1H, m), 7.22-7.30 (5H, m), 7.34 (1H, s), 7.73 (1H, t, J=5.6Hz), 7.83 (1H, d, J=10.7 Hz), 8.08 (1H, t, J=5.6 Hz), 8.15 (1H, d, J=7.8Hz), 8.30 (2H, dt, J=18.7, 5.7 Hz), 8.49 (1H, d, J=8.8 Hz).

MS (APCI) m/z: 990 (M+H)⁺

Process 2: Antibody-Drug Conjugate (4)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm⁻¹ was used). The solution (1 mL) was placed in a 1.5 mL polypropylenetube and charged with an aqueous solution of 10 mM TCEP (0.0155 mL; 2.3equivalents per antibody molecule) and an aqueous solution of 1 Mdipotassium hydrogen phosphate (0.050 mL). After confirming that thesolution had a pH of 7.4±0.1, the disulfide bond at the hinge part inthe antibody was reduced by incubating at 37° C. for 1 hour. Conjugationbetween antibody and drug linker: After adding DMSO (0.072 mL) and aDMSO solution containing 10 mM of the compound of Process 3 of Example 2(0.031 mL; 4.6 equivalents per antibody molecule) to the above solutionat room temperature, it was stirred by using a tube rotator forconjugating the drug linker to the antibody at room temperature for 40minutes. Next, an aqueous solution (0.0078 mL; 9.2 equivalents perantibody molecule) of 100 mM NAC was added thereto and stirred at roomtemperature to terminate the reaction of the drug linker for another 20minutes.

Purification: The above solution was subjected to purification using theCommon procedure D (ABS was used as buffer solution) to yield 6 mL of asolution containing the compound of interest. By using the Commonprocedure E, the following characteristic values were obtained.

Antibody concentration: 1.32 mg/mL, antibody yield: 7.9 mg (79%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.1.

Example 5 Antibody-Drug Conjugate (5)

Process 1: Antibody-Drug Conjugate (5)

The amount of the aqueous solution of 10 mM TCEP was adjusted such thatthe molar ratio of TCEP to the antibody at the antibody reduction was4.6. And the amount of the 10 mM drug linker solution added was adjustedsuch that the molar ratio of the compound of Process 1 of Example 4 tothe antibody at the drug linker conjugation was 9.2. Then the amount ofthe aqueous solution of 100 mM NAC added was adjusted such that themolar ratio of NAC to the antibody at the termination of the reactionwas 18.4. By the same procedures as Process 2 of Example 4, 6 mL of asolution containing the titled antibody-drug conjugate was obtained, andthe following characteristic values were obtained.

Antibody concentration: 1.23 mg/mL, antibody yield: 7.4 mg (74%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.1.

Example 6 Antibody-Drug Conjugate (6)

Process 1:N-{3-[2-(2-{[3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]propanoyl}glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (100 mg, 0.119 mmol) obtained in Process 2 of Example 2 wasreacted in the same manner as Process 3 of Example 2 by usingdiisopropylethylamine (20.8 μL, 0.119 mmol) instead of triethylamine andN-succinimidyl3-(2-(2-(3-maleinimidepropanamide)ethoxy)ethoxy)propanoate (50.7 mg,0.119 mmol) instead of N-succinimidyl 6-maleimide hexanoate to yield thetitled compound as a pale yellow solid (66.5 mg, 48%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.85 (3H, t, J=7.4 Hz), 1.65-1.74 (2H, m),1.77-1.90 (2H, m), 2.07-2.19 (4H, m), 2.30 (2H, t, J=7.2 Hz), 2.33-2.36(2H, m), 2.38 (3H, s), 2.76 (1H, dd, J=13.7, 9.8 Hz), 2.96-3.18 (9H, m),3.42-3.44 (4H, m), 3.53-3.76 (10H, m), 4.43 (1H, td, J=8.6, 4.7 Hz),5.14 (1H, d, J=18.8 Hz), 5.23 (1H, d, J=18.8 Hz), 5.38 (1H, d, J=17.2Hz), 5.42 (1H, d, J=17.2 Hz), 5.52-5.58 (1H, m), 6.52 (1H, s), 6.98 (2H,s), 7.12-7.17 (1H, m), 7.18-7.25 (4H, m), 7.29 (1H, s), 7.69 (1H, t,J=5.5 Hz), 7.78 (1H, d, J=11.3 Hz), 7.98-8.03 (2H, m), 8.11 (1H, d,J=7.8 Hz), 8.16 (1H, t, J=5.7 Hz), 8.23 (1H, t, J=5.9 Hz), 8.44 (1H, d,J=9.0 Hz).

MS (APCI) m/z: 1149 (M+H)⁺

Process 2: Antibody-Drug Conjugate (6)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm⁻¹ was used). The solution (1.25 mL) was placed in a 1.5 mLpolypropylene tube and charged with an aqueous solution of 10 mM TCEP(0.019 mL; 2.3 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (0.0625 mL). Afterconfirming that the solution had a pH of 7.4±0.1, the disulfide bond atthe hinge part in the antibody was reduced by incubating at 37° C. for 1hour. Conjugation between antibody and drug linker: After adding DMSO(Sigma-Aldrich Co. LLC; 0.109 mL) and a DMSO solution containing 10 mMof the compound of Process 1 (0.039 mL; 4.6 equivalents per antibodymolecule) to the above solution at room temperature, it was stirred byusing a tube rotator for conjugating the drug linker to the antibody atroom temperature for 40 minutes. Next, an aqueous solution (0.008 mL) of100 mM NAC was added thereto and stirred at room temperature toterminate the reaction of the drug linker for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D (ABS was used as buffer solution) described inProduction method 1 to yield 6 mL of a solution containing the compoundof interest.

Physicochemical characterization: By using the Common procedure E, thefollowing characteristic values were obtained.

Antibody concentration: 1.76 mg/mL, antibody yield: 10.6 mg (85%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.6.

Example 7 Antibody-Drug Conjugate (7)

Process 1: Antibody-Drug Conjugate (7)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm¹ was used). The solution (1.25 mL) was placed in a 1.5 mLpolypropylene tube and charged with an aqueous solution of 10 mM TCEP(0.039 mL; 4.6 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (0.0625 mL). Afterconfirming that the solution had a pH of 7.4±0.1, the disulfide bond atthe hinge part in the antibody was reduced by incubating at 37° C. for 1hour. Conjugation between antibody and drug linker: After adding DMSO(0.072 mL) and a DMSO solution containing 10 mM of the compound ofProcess 1 of Example 6 (0.078 mL; 9.2 equivalents per antibody molecule)to the above solution at room temperature, it was stirred by using atube rotator for conjugating the drug linker to the antibody at roomtemperature for 40 minutes. Next, an aqueous solution (0.0155 mL) of 100mM NAC was added thereto and stirred at room temperature to terminatethe reaction of the drug linker for another 20 minutes. Purification:The above solution was subjected to purification using the Commonprocedure D (ABS was used as buffer solution) to yield 6 mL of asolution containing the compound of interest.

Physicochemical characterization: By using the Common procedure E, thefollowing characteristic values were obtained.

Antibody concentration: 1.93 mg/mL, antibody yield: 11.6 mg (93%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.9.

Example 8 Antibody-Drug Conjugate (8)

Process 1: Antibody-Drug Conjugate (8)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm⁻¹ was used). The solution (1.25 mL) was placed in a 1.5 mLpolypropylene tube and charged with an aqueous solution of 10 mM TCEP(0.039 mL; 4.6 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (0.0625 mL). Afterconfirming that the solution had a pH of 7.4±0.1, the disulfide bond atthe hinge part in the antibody was reduced by incubating at 37° C. for 1hour. Conjugation between antibody and drug linker: After adding DMSO(0.072 mL) and a DMSO solution containing 10 mM of the compound ofProcess 1 of Example 6 (0.078 mL; 9.2 equivalents per antibody molecule)to the above solution at room temperature, it was stirred by using atube rotator for conjugating the drug linker to the antibody at roomtemperature for 40 minutes. Next, an aqueous solution (0.0155 mL) of 100mM NAC was added thereto and stirred at room temperature to terminatethe reaction of the drug linker for another 20 minutes. Purification:The above solution was subjected to purification using the Commonprocedure D-1 (ABS was used as buffer solution) to yield 5.7 mL of asolution containing the compound of interest.

Physicochemical characterization: By using the Common procedure E, thefollowing characteristic values were obtained.

Antibody concentration: 1.50 mg/mL, antibody yield: 8.55 mg (86%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.2.

Example 9 Antibody-Drug Conjugate (9)

Process 1:N-[19-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxo-16-azanonadecan-1-oyl]glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (90 mg, 0.107 mmol) obtained in Process 2 of Example 2 wasreacted in the same manner as Process 3 of Example 2 by usingdiisopropylethylamine (18.7 μL, 0.107 mmol) instead of triethylamine andN-succinimidyl1-maleinimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oate (55.1 mg,0.107 mmol) instead of N-succinimidyl 6-maleimide hexanoate to yield thetitled compound as a pale yellow solid (50 mg, 37%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.85 (3H, t, J=7.2 Hz), 1.64-1.74 (2H, m),1.77-1.90 (2H, m), 2.06-2.19 (4H, m), 2.27-2.32 (2H, m), 2.33-2.37 (2H,m), 2.38 (3H, s), 2.72-2.80 (3H, m), 2.96-3.19 (6H, m), 3.39-3.48 (10H,m), 3.52-3.75 (10H, m), 4.39-4.48 (1H, m), 5.14 (1H, d, J=18.8 Hz), 5.23(1H, d, J=18.8 Hz), 5.38 (1H, d, J=17.0 Hz), 5.42 (1H, d, J=17.0 Hz),5.52-5.58 (1H, m), 6.52 (1H, s), 6.98 (1H, s), 7.13-7.24 (5H, m), 7.29(1H, s), 7.69 (1H, t, J=5.5 Hz), 7.78 (1H, d, J=10.9 Hz), 7.98-8.03 (2H,m), 8.10 (1H, d, J=7.8 Hz), 8.16 (1H, t, J=5.7 Hz), 8.23 (1H, t, J=5.7Hz), 8.44 (1H, d, J=8.6 Hz).

MS (APCI) m/z: 1237 (M+H)⁺

Process 2: Antibody-Drug Conjugate (9)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1, the titled antibody-drug conjugate wasobtained in the same manner as Process 2 of Example 6.

Antibody concentration: 1.75 mg/mL, antibody yield: 10.5 mg (84%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.4.

Example 10 Antibody-Drug Conjugate (10)

Process 1: Antibody-Drug Conjugate (10)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1 of Example 9, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 7.Antibody concentration: 1.79 mg/mL, antibody yield: 10.7 mg (86%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.0.

Example 11 Intermediate (11)

Process 1:tert-Butyl[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)ethyl]carbamate

Methanesulfonic acid salt of exatecan (3.10 g, 5.47 mol) was reacted inthe same manner as Process 1 of Example 1 by using{2-[(tert-butoxycarbonyl)amino]ethoxy}acetic acid (J. Med. Chem., 1992,vol. 35, pp. 292; 1.55 g, 6.01 mmol) instead of4-(tert-butoxycarbonylamino)butanoic acid to yield the titled compoundas a pale yellow solid (2.56 g, 73%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.3 Hz), 1.26 (9H, s),1.81-1.91 (2H, m), 2.13-2.22 (2H, m), 2.40 (3H, s), 3.08-3.26 (4H, m),3.43-3.53 (2H, m), 4.00 (1H, d, J=15.1 Hz), 4.05 (1H, d, J=15.1 Hz),5.14 (1H, d, J=18.7 Hz), 5.22 (1H, d, J=18.7 Hz), 5.40 (1H, d, J=16.6Hz), 5.44 (1H, d, J=16.6 Hz), 5.59-5.66 (1H, m), 6.53 (1H, s), 6.86 (1H,t, J=5.4 Hz), 7.31 (1H, s), 7.79 (1H, d, J=10.9 Hz), 8.49 (1H, d, J=9.1Hz).

MS (APCI) m/z: 637 (M+H)⁺

Process 2:2-(2-Aminoethoxy)-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]acetamide

The compound (1.50 g, 2.36 mol) obtained in Process 1 above was reactedin the same manner as Process 2 of Example 1 to yield trifluoroaceticacid salt of the titled compound as a pale yellow solid (1.50 g,quantitative).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.5 Hz), 1.81-1.92 (2H, m),2.15-2.23 (2H, m), 2.41 (3H, s), 3.05 (2H, t, J=5.1 Hz), 3.15-3.23 (2H,m), 3.71 (2H, t, J=5.1 Hz), 4.10 (2H, s), 5.19 (1H, d, J=18.7 Hz), 5.24(1H, d, J=18.7 Hz), 5.43 (2H, s), 5.58-5.66 (1H, m), 6.55 (1H, s), 7.33(1H, s), 7.73-7.84 (4H, m), 8.55 (1H, d, J=9.1 Hz).

MS (APCI) m/z: 537 (M+H)⁺

Example 12 Antibody-Drug Conjugate (12)

Process 1:N-(tert-Butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)ethyl]glycinamide

The compound (554 mg, 0.85 mmol) of Process 2 of Example 11 was reactedin the same manner as Process 1 of Example 2 to yield the titledcompound (775 mg, 95%). ¹H-NMR (400 MHz, DMSO-d₆) δ: 0.85 (3H, t, J=7.3Hz), 1.36 (9H, s), 1.78-1.89 (2H, m), 2.13-2.22 (2H, m), 2.39 (3H, s),2.71 (1H, dd, J=13.4, 9.8 Hz), 2.95 (1H, dd, J=13.4, 4.3 Hz), 3.09-3.23(1H, m), 3.23-3.32 (2H, m), 3.40-3.62 (8H, m), 3.73 (1H, dd, J=16.5, 5.5Hz), 4.03 (2H, s), 4.39-4.47 (1H, m), 5.17 (1H, d, J=18.9 Hz), 5.25 (1H,d, J=18.9 Hz), 5.41 (1H, d, J=16.8 Hz), 5.45 (1H, d, J=16.8 Hz),5.57-5.64 (1H, m), 6.54 (1H, s), 6.99 (1H, t, J=5.8 Hz), 7.13-7.26 (5H,m), 7.31 (1H, s), 7.76-7.82 (2H, m), 7.90 (1H, t, J=5.2 Hz), 8.13 (1H,d, J=7.9 Hz), 8.27 (1H, t, J=5.8 Hz), 8.49 (1H, d, J=8.5 Hz).

MS (APCI) m/z: 955 (M+H)⁺

Process 2:Glycylglycyl-L-phenylalanyl-N-[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)ethyl]glycinamide

The compound (630 mg, 0.659 mmol) obtained in Process 1 above wasreacted in the same manner as Process 2 of Example 2 to yieldtrifluoroacetic acid salt of the titled compound (588 mg, 92%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.3 Hz), 1.79-1.90 (2H, m),2.13-2.22 (2H, m), 2.39 (3H, s), 2.71 (1H, dd, J=13.4, 10.1 Hz), 2.99(1H, dd, J=13.4, 4.3 Hz), 3.09-3.23 (1H, m), 3.24-3.32 (3H, m),3.41-3.71 (7H, m), 3.86 (1H, dd, J=16.8, 5.8 Hz), 4.04 (2H, s), 4.52(1H, td, J=9.0, 4.1 Hz), 5.17 (1H, d, J=18.9 Hz), 5.25 (1H, d, J=18.9Hz), 5.41 (1H, d, J=16.5 Hz), 5.45 (1H, d, J=16.5 Hz), 5.56-5.65 (1H,m), 6.55 (1H, s), 7.13-7.26 (5H, m), 7.32 (1H, s), 7.80 (1H, d, J=11.0Hz), 7.87-8.01 (4H, m), 8.29-8.36 (2H, m), 8.46-8.55 (2H, m).

MS (APCI) m/z: 855 (M+H)⁺

Process 3:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)ethyl]glycinamide

The compound (240 mg, 0.247 mmol) obtained in Process 2 above wasreacted in the same manner as Process 3 of Example 2 to yield the titledcompound (162 mg, 62%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.6 Hz), 1.13-1.22 (2H, m),1.40-1.51 (4H, m), 1.78-1.90 (2H, m), 2.09 (2H, t, J=7.6 Hz), 2.14-2.21(2H, m), 2.39 (3H, s), 2.74 (1H, dd, J=13.6, 9.7 Hz), 2.96 (1H, dd,J=13.6, 4.5 Hz), 3.08-3.24 (1H, m), 3.24-3.30 (1H, m), 3.33-3.40 (4H,m), 3.47-3.68 (7H, m), 3.72 (1H, dd, J=16.6, 5.7 Hz), 4.03 (2H, s), 4.42(1H, td, J=8.6, 4.2 Hz), 5.17 (1H, d, J=18.7 Hz), 5.25 (1H, d, J=18.7Hz), 5.40 (1H, d, J=17.2 Hz), 5.44 (1H, d, J=17.2 Hz), 5.57-5.64 (1H,m), 6.52 (1H, s), 6.99 (2H, s), 7.13-7.25 (5H, m), 7.31 (1H, s),7.74-7.81 (2H, m), 7.99 (1H, t, J=5.7 Hz), 8.03-8.11 (2H, m), 8.22 (1H,t, J=5.7 Hz), 8.47 (1H, d, J=9.1 Hz).

MS (APCI) m/z: 1048 (M+H)⁺

Process 4: Antibody-Drug Conjugate (12)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 3, the titled antibody-drug conjugate wasobtained in the same manner as Process 2 of Example 6. The solution wasfurther concentrated by the Common procedure A. After that, by using theCommon procedure E, the following characteristic values were obtained.

Antibody concentration: 10.77 mg/mL, antibody yield: 7.5 mg (60%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.7.

Example 13 Antibody-Drug Conjugate (13)

Process 1: Antibody-Drug Conjugate (13)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 3 of Example 12, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 7. Thesolution was further concentrated by the Common procedure A. After that,by using the Common procedure E, the following characteristic valueswere obtained. Antibody concentration: 10.69 mg/mL, antibody yield: 7.5mg (60%), and average number of conjugated drug molecules (n) perantibody molecule: 6.9.

Example 14 Intermediate (14)

Process 1: tert-Butyl(3-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-3-oxopropyl)carbamate

Methanesulfonic acid salt of exatecan (500 mg, 0.941 mmol) was reactedin the same manner as Process 1 of Example 1 by usingN-(tert-butoxycarbonyl)-3-alanine instead of4-(tert-butoxycarbonylamino)butanoic acid to yield the titled compoundas a yellow-brown solid (616 mg, quantitative).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.2 Hz), 1.29 (9H, s), 1.86(2H, dt, J=15.1, 7.3 Hz), 2.04-2.22 (2H, m), 2.31 (2H, t, J=6.8 Hz),2.40 (3H, s), 3.10-3.26 (4H, m), 5.15 (1H, d, J=18.8 Hz), 5.26 (1H, d,J=19.2 Hz), 5.42 (2H, dd, J=18.8, 16.4 Hz), 5.57 (1H, dt, J=8.5, 4.2Hz), 6.53 (1H, s), 6.78 (1H, t, J=5.5 Hz), 7.30 (1H, s), 7.80 (1H, d,J=11.0 Hz), 8.46 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 607 (M+H)⁺

Process 2:N-[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound obtained in Process 1 above was reacted in the same manneras Process 2 of Example 1 to yield trifluoroacetic acid salt of thetitled compound as a yellow solid (499 mg, 86%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.2 Hz), 1.86 (2H, dquin,J=14.6, 7.2, 7.2, 7.2, 7.2 Hz), 2.06-2.27 (1H, m), 2.41 (3H, s),2.46-2.57 (2H, m), 3.08 (2H, t, J=6.8 Hz), 3.14-3.24 (2H, m), 5.22 (1H,d, J=18.8 Hz), 5.29 (1H, d, J=18.8 Hz), 5.43 (2H, s), 5.58 (1H, dt,J=8.5, 4.5 Hz), 6.55 (1H, s), 7.32 (1H, s), 7.74 (3H, brs), 7.82 (1H, d,J=11.0 Hz), 8.67 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 507 (M+H)⁺

Example 15 Antibody-Drug Conjugate (15)

Process 1:N-(tert-Butoxycarbonyl)glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound (484 mg, 0.780 mmol) obtained in Process 2 of Example 14was reacted in the same manner as Process 1 of Example 2 to yield thetitled compound as a pale yellow solid (626 mg, 87%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.27-1.42 (9H, m),1.77-1.93 (2H, m), 2.06-2.22 (2H, m), 2.36 (2H, t, J=7.2 Hz), 2.40 (3H,d, J=1.6 Hz), 2.44-2.54 (2H, m), 2.76 (1H, dd, J=14.5, 10.2 Hz), 3.02(1H, dd, J=13.9, 4.5 Hz), 3.12-3.22 (2H, m), 3.52 (6H, d, J=6.3 Hz),4.42-4.54 (1H, m), 5.19 (1H, d, J=19.2 Hz), 5.26 (1H, d, J=18.4 Hz),5.42 (1H, dd, J=18.4, 16.4 Hz), 5.57 (1H, dt, J=8.7, 4.4 Hz), 6.53 (1H,s), 6.98 (1H, t, J=5.9 Hz), 7.14-7.28 (5H, m), 7.31 (1H, s), 7.77-7.84(1H, m), 7.91 (1H, t, J=5.5 Hz), 8.16 (1H, d, J=7.8 Hz), 8.27 (1H, t,J=5.1 Hz), 8.52 (1H, d, J=9.0 Hz).

Process 2:Glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamidetrifluoroacetate

The compound (624 mg, 0.675 mmol) obtained in Process 1 above wasreacted in the same manner as Process 2 of Example 2 to yield the titledcompound as a yellow solid (626 mg, 92%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.86 (2H, tt,J=14.5, 7.2 Hz), 2.07-2.22 (2H, m), 2.36 (2H, t, J=7.2 Hz), 2.40 (3H,s), 2.44-2.54 (2H, m), 2.75 (1H, dd, J=13.7, 9.8 Hz), 3.04 (1H, dd,J=13.7, 4.3 Hz), 3.12-3.22 (2H, m), 3.58 (2H, d, J=4.7 Hz), 3.69 (3H,td, J=11.2, 5.7 Hz), 3.87 (1H, dd, J=17.0, 5.7 Hz), 4.54 (1H, m, J=17.8,4.5 Hz), 5.19 (1H, d, J=19.2 Hz), 5.26 (1H, d, J=18.8 Hz), 5.43 (2H, s),5.51-5.60 (1H, m), 6.55 (1H, s), 7.14-7.29 (5H, m), 7.32 (1H, s), 7.81(1H, d, J=10.9 Hz), 7.88 (1H, t, J=5.7 Hz), 7.97 (3H, brs), 8.29-8.38(2H, m), 8.50 (1H, t, J=5.7 Hz), 8.55 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 825 (M+H)⁺

Process 3:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 above wasreacted in the same manner as Process 3 of Example 2 to yield the titledcompound as a solid (14.0 mg, 21%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.2 Hz), 1.12-1.22 (2H, m),1.39-1.51 (4H, m), 1.79-1.91 (2H, m), 2.02-2.20 (2H, m), 2.07 (2H, t,J=7.4 Hz), 2.30-2.42 (4H, m), 2.40 (3H, s), 2.78 (1H, dd, J=14.1, 9.4Hz), 3.02 (1H, dd, J=14.7, 4.9 Hz), 3.12-3.21 (2H, m), 3.26-3.42 (2H,m), 3.50-3.80 (6H, m), 4.40-4.51 (1H, m), 5.19 (1H, d, J=19.6 Hz), 5.26(1H, d, J=19.2 Hz), 5.42 (2H, brs), 5.51-5.62 (1H, m), 6.53 (1H, s),6.99 (2H, s), 7.13-7.28 (5H, m), 7.31 (1H, s), 7.74-7.84 (2H, m), 8.01(1H, t, J=5.3 Hz), 8.06 (1H, t, J=5.7 Hz), 8.14 (1H, d, J=8.2 Hz), 8.25(1H, t, J=5.7 Hz), 8.53 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 1018 (M+H)⁺

Process 4: Antibody-Drug Conjugate (15)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure C-1 and Common procedure B (asabsorption coefficient at 280 nm, 1.37 mLmg⁻¹ cm⁻¹ was used). Thesolution (1.0 mL) was collected into a 2 mL tube and charged with anaqueous solution of 10 mM TCEP (0.0155 mL; 2.3 equivalents per antibodymolecule) and an aqueous solution of 1 M dipotassium hydrogen phosphate(0.050 mL). After confirming that the solution had a pH of 7.4±0.1, thedisulfide bond at the hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating thesolution at 22° C. for 10 minutes, a DMSO solution (0.0311 mL; 4.6equivalents per antibody molecule) containing 10 mM of the compoundobtained in Process 3 was added thereto and incubated for conjugatingthe drug linker to the antibody at 22° C. for 40 minutes. Next, anaqueous solution (0.00622 mL; 9.2 equivalents per antibody molecule) of100 mM NAC was added thereto and incubated at 22° C. to terminate thereaction of the drug linker for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS6.0 was used as buffer solution) to yield 6 mLof a solution containing the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E, thefollowing characteristic values were obtained.

Antibody concentration: 1.18 mg/mL, antibody yield: 7.08 mg (71%), andaverage number of conjugated drug molecules (n) per antibody molecule:2.0.

Example 16 Antibody-Drug Conjugate (16)

Process 1: Antibody-Drug Conjugate (16)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure C-1 and Common procedure B (asabsorption coefficient at 280 nm, 1.37 mLmg⁻¹ cm⁻¹ was used). Thesolution (1.0 mL) was collected into a 2 mL tube and charged with anaqueous solution of 10 mM TCEP (0.0311 mL; 4.6 equivalents per antibodymolecule) and an aqueous solution of 1 M dipotassium hydrogen phosphate(0.050 mL). After confirming that the solution had a pH of 7.4±0.1, thedisulfide bond at the hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating thesolution at 22° C. for 10 minutes, a DMSO solution (0.0622 mL; 9.2equivalents per antibody molecule) containing 10 mM of the compoundobtained in Process 3 of Example 15 was added thereto and incubated forconjugating the drug linker to the antibody at 22° C. for 40 minutes.Next, an aqueous solution (0.0124 mL; 18.4 equivalents per antibodymolecule) of 100 mM NAC was added thereto and incubated at 22° C. toterminate the reaction of the drug linker for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS6.0 was used as buffer solution) to yield 6 mLof a solution containing the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E, thefollowing characteristic values were obtained.

Antibody concentration: 1.03 mg/mL, antibody yield: 6.18 mg (62%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.8.

Example 17 Antibody-Drug Conjugate (17)

Process 1:N-[3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 15was reacted in the same manner as Process 3 of Example 2 by usingN-succinimidyl 3-maleimide propionate instead of N-succinimidyl6-maleimide hexanoate to yield the titled compound as a pale yellowsolid (36.0 mg, 57%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.4 Hz), 1.85 (2H, dt,J=14.4, 7.5 Hz), 2.05-2.22 (2H, m), 2.40 (3H, s), 2.30-2.44 (5H, m),2.73-2.84 (1H, m), 3.02 (1H, dd, J=13.9, 4.5 Hz), 3.17 (3H, d, J=5.1Hz), 3.26-3.40 (2H, m), 3.41-3.81 (6H, m), 4.40-4.51 (1H, m), 5.19 (1H,d, J=19.2 Hz), 5.26 (1H, d, J=18.8 Hz), 5.42 (2H, brs), 5.52-5.61 (1H,m), 6.53 (1H, s), 6.99 (2H, s), 7.13-7.28 (5H, m), 7.31 (1H, s), 7.80(2H, d, J=10.2 Hz), 8.03 (1H, t, J=5.5 Hz), 8.12 (1H, d, J=8.2 Hz),8.20-8.31 (2H, m), 8.52 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 976 (M+H)⁺

Process 2: Antibody-Drug Conjugate (17)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 2 of Example 6.

Antibody concentration: 1.74 mg/mL, antibody yield: 10.4 mg (83%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.7.

Example 18 Antibody-Drug Conjugate (18)

Process 1: Antibody-Drug Conjugate (18)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1 of Example 17, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 7.Antibody concentration: 1.98 mg/mL, antibody yield: 11.9 mg (95%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.6.

Example 19 Antibody-Drug Conjugate (19)

Process 1:N-{3-[2-(2-{[3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino})ethoxy]propanoyl}glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 15was reacted in the same manner as Process 3 of Example 2 by usingN-succinimidyl3-(2-(2-(3-maleinimidepropanamide)ethoxy)ethoxy)propanoate instead ofN-succinimidyl 6-maleimide hexanoate to yield the titled compound as asolid (23.0 mg, 31%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.4 Hz), 1.77-1.92 (2H, m),2.07-2.21 (2H, m), 2.27-2.42 (6H, m), 2.40 (3H, s), 2.74-2.84 (1H, m),2.97-3.06 (1H, m), 3.09-3.21 (4H, m), 3.25-3.39 (6H, m), 3.45 (4H, s),3.50-3.80 (8H, m), 4.41-4.51 (1H, m), 5.19 (1H, d, J=18.4 Hz), 5.26 (1H,m, J=18.4 Hz), 5.42 (2H, brs), 5.51-5.61 (1H, m), 6.54 (1H, s), 7.00(2H, s), 7.13-7.28 (5H, m), 7.31 (1H, s), 7.74-7.87 (2H, m), 7.93-8.07(2H, m), 8.09-8.21 (2H, m), 8.26 (1H, brs), 8.54 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 1135 (M+H)⁺

Process 2: Antibody-Drug Conjugate (19)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 2 of Example 6.

Antibody concentration: 1.60 mg/mL, antibody yield: 9.6 mg (77%), andaverage number of conjugated drug molecules (n) per antibody molecule:1.9.

Example 20 Antibody-Drug Conjugate (20)

Process 1: Antibody-Drug Conjugate (20)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1 of Example 19, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 7.Antibody concentration: 1.69 mg/mL, antibody yield: 10.1 mg (81%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.0.

Example 21 Antibody-Drug Conjugate (21)

Process 1:N-[19-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 15was reacted in the same manner as Process 3 of Example 2 by usingN-succinimidyl1-maleinimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoate instead ofN-succinimidyl 6-maleimide hexanoate to yield the titled compound as asolid (23.0 mg, 29%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.0 Hz), 1.85 (2H, tt,J=14.6, 7.1 Hz), 2.06-2.22 (2H, m), 2.40 (3H, s), 2.28-2.43 (6H, m),2.78 (1H, dd, J=13.7, 9.4 Hz), 3.02 (1H, dd, J=14.1, 3.9 Hz), 3.09-3.22(4H, m), 3.27-3.41 (4H, m), 3.47 (12H, d, J=8.6 Hz), 3.53-3.81 (10H, m),4.41-4.51 (1H, m), 5.19 (1H, d, J=19.2 Hz), 5.26 (1H, d, J=18.8 Hz),5.42 (2H, brs), 5.53-5.61 (1H, m), 6.54 (1H, s), 7.00 (2H, s), 7.12-7.29(5H, m), 7.31 (1H, s), 7.74-7.85 (2H, m), 8.03 (2H, d, J=6.6 Hz),8.11-8.21 (2H, m), 8.27 (1H, t, J=5.9 Hz), 8.54 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 1224 (M+H)⁺

Process 2: Antibody-Drug Conjugate (21)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1, the titled antibody-drug conjugate wasobtained in the same manner as Process 2 of Example 6.

Antibody concentration: 1.77 mg/mL, antibody yield: 10.6 mg (85%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.2.

Example 22 Antibody-Drug Conjugate (22)

Process 1: Antibody-Drug Conjugate (22)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1 of Example 21, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 7.Antibody concentration: 1.89 mg/mL, antibody yield: 11.3 mg (90%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.2.

Example 23 Intermediate (23)

Process 1: tert-Butyl(6-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-6-oxohexyl)carbamate

Methanesulfonic acid salt of exatecan (0.500 g, 0.882 mmol) was reactedin the same manner as Process 1 of Example 1 by using6-(tert-butoxycarbonylamino)hexanoic acid instead of4-(tert-butoxycarbonylamino)butanoic acid to yield the titled compound(0.620 g, quantitative).

¹H-NMR (DMSO-d₆) δ: 0.83 (3H, t, J=7.8 Hz), 1.14-1.28 (2H, m), 1.31 (9H,s), 1.47-1.61 (2H, m), 1.75-1.89 (2H, m), 2.04-2.17 (4H, m), 2.35 (3H,s), 2.81-2.88 (2H, m), 3.09-3.16 (2H, m), 5.10 (1H, d, J=19.4 Hz), 5.16(1H, d, J=19.4 Hz), 5.39 (2H, s), 5.48-5.55 (1H, m), 6.50 (1H, s),6.73-6.78 (1H, m), 7.26 (1H, s), 7.74 (1H, d, J=10.9 Hz), 8.39 (1H, d,J=9.0 Hz).

Process 2:6-Amino-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]hexanamide

The compound (0.397 g, 0.611 mmol) obtained in Process 1 above wasreacted in the same manner as Process 2 of Example 1 to yieldtrifluoroacetic acid salt of the titled compound (0.342 g, 84%).

¹H-NMR (DMSO-d₆) δ: 0.88 (3H, t, J=7.2 Hz), 1.31-1.41 (2H, m), 1.52-1.70(4H, m), 1.80-1.94 (2H, m), 2.05-2.18 (2H, m), 2.21 (2H, t, J=7.4 Hz),2.40 (3H, s), 2.81 (2H, t, J=7.4 Hz), 3.10-3.25 (2H, m), 3.33 (2H, brs),5.18 (1H, d, J=19.8 Hz), 5.22 (1H, d, J=19.8 Hz), 5.41 (2H, d, J=16.6Hz), 5.45 (2H, d, J=16.6 Hz), 5.53-5.60 (1H, m), 6.55 (1H, s), 7.32 (1H,s), 7.80 (1H, d, J=10.9 Hz), 8.49 (1H, d, J=9.2 Hz).

Example 24 Antibody-Drug Conjugate (24)

Process 1:N-(tert-Butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-(6-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-6-oxohexyl)glycinamide

The compound (0.170 g, 0.516 mmol) obtained in Process 2 of Example 23was reacted in the same manner as Process 1 of Example 2 to yield thetitled compound (0.225 g, 91%).

¹H-NMR (DMSO-d₆) δ: 0.88 (3H, t, J=7.4 Hz), 1.43-1.70 (6H, m), 1.87 (2H,td, J=15.0, 7.4 Hz), 2.10-2.22 (3H, m), 2.28-2.37 (1H, m), 2.42 (3H, s),2.78-2.85 (1H, m), 3.01-3.10 (3H, m), 3.15-3.22 (2H, m), 3.54-3.61 (5H,m), 3.62-3.69 (1H, m), 4.44-4.53 (1H, m), 5.17 (1H, d, J=19.2 Hz), 5.25(1H, d, J=19.2 Hz), 5.45 (2H, s), 5.54-5.61 (1H, m), 6.55 (1H, s), 7.02(1H, t, J=6.1 Hz), 7.11-7.28 (5H, m), 7.33 (1H, s), 7.63-7.69 (1H, m),7.82 (1H, d, J=11.0 Hz), 7.90-7.96 (1H, m), 8.17 (1H, d, J=7.8 Hz), 8.28(1H, t, J=5.5 Hz), 8.46 (1H, d, J=9.0 Hz).

Process 2:Glycylglycyl-L-phenylalanyl-N-(6-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-6-oxohexyl)glycinamide

The compound (0.105 g, 0.108 mmol) obtained in Process 1 above wasreacted in the same manner as Process 2 of Example 2 to yield the titledcompound (0.068 mg, 65%).

¹H-NMR (DMSO-d₆) δ: 0.89 (3H, t, J=7.4 Hz), 1.15-1.67 (6H, m), 1.79-1.97(2H, m), 2.08-2.24 (4H, m), 2.42 (3H, s), 2.76-2.82 (1H, m), 3.00-3.10(5H, m), 3.19 (1H, s), 3.50-3.63 (2H, m), 3.64-3.76 (3H, m), 3.84-3.92(1H, m), 4.51-4.59 (1H, m), 5.17 (1H, d, J=19.4 Hz), 5.24 (1H, d, J=19.4Hz), 5.44 (2H, s), 5.53-5.61 (1H, m), 6.55 (1H, brs), 7.15-7.29 (5H, m),7.33 (1H, s), 7.72-7.78 (1H, m), 7.82 (1H, d, J=11.0 Hz), 7.96-8.08 (2H,m), 8.30-8.38 (2H, m), 8.46-8.56 (2H, m).

Process 3:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(6-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-6-oxohexyl)glycinamide

The compound (58 mg, 0.060 mmol) obtained in Process 2 above was reactedin the same manner as Process 3 of Example 2 to yield the titledcompound (39 mg, 62%).

¹H-NMR (CD₃OD) δ: 0.99 (3H, t, J=7.4 Hz), 1.27 (2H, td, J=11.6, 6.1 Hz),1.38-1.44 (2H, m), 1.50-1.63 (6H, m), 1.65-1.80 (2H, m), 1.89-1.98 (2H,m), 2.17-2.25 (3H, m), 2.26-2.36 (3H, m), 2.40 (3H, s), 2.95 (1H, dd,J=14.3, 9.2 Hz), 3.12 (1H, dd, J=13.7, 5.7 Hz), 3.15-3.25 (4H, m), 3.44(2H, t, J=7.2 Hz), 3.65 (1H, d, J=17.2 Hz), 3.76 (1H, d, J=17.2 Hz),3.79-3.86 (4H, m), 4.43 (1H, dd, J=8.9, 6.0 Hz), 5.10 (1H, d, J=18.9Hz), 5.25 (1H, d, J=18.9 Hz), 5.35 (1H, d, J=16.6 Hz), 5.56 (1H, d,J=16.0 Hz), 5.60-5.64 (1H, m), 6.76 (2H, s), 7.12-7.24 (6H, m), 7.58(1H, s), 7.60 (1H, d, J=10.9 Hz), 7.68 (1H, t, J=5.7 Hz).

MS (ESI) m/z: 1060 (M+H)⁺

Process 4: Antibody-Drug Conjugate (24)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure C-1 and Common procedure B (asabsorption coefficient at 280 nm, 1.37 mLmg⁻¹ cm⁻¹ was used). Thesolution (9.0 mL) was collected into a 50 mL tube and charged with anaqueous solution of 10 mM TCEP (0.140 mL; 2.3 equivalents per antibodymolecule) and an aqueous solution of 1 M dipotassium hydrogen phosphate(0.450 mL). After confirming that the solution had a pH of 7.4±0.1, thedisulfide bond at the hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating thesolution at 22° C. for 10 minutes, a DMSO solution (0.280 mL; 4.6equivalents per antibody molecule) containing 10 mM of the compound inProcess 3 was added thereto and incubated for conjugating the druglinker to the antibody at 22° C. for 40 minutes. Next, an aqueoussolution (0.0559 mL; 9.2 equivalents per antibody molecule) of 100 mMNAC was added thereto and incubated at 22° C. to terminate the reactionof the drug linker for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS7.4 was used as buffer solution) to yield asolution containing the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E, thefollowing characteristic values were obtained.

Antibody concentration: 3.30 mg/mL, antibody yield: 53.5 mg (59%), andaverage number of conjugated drug molecules (n) per antibody molecule:1.7.

Example 25 Antibody-Drug Conjugate (25)

Process 1: Antibody-Drug Conjugate (25)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure C-1 and Common procedure B (asabsorption coefficient at 280 nm, 1.37 mLmg⁻¹ cm⁻¹ was used). Thesolution (9.0 mL) was collected into a 50 mL tube and charged with anaqueous solution of 10 mM TCEP (0.280 mL; 4.6 equivalents per antibodymolecule) and an aqueous solution of 1 M dipotassium hydrogen phosphate(0.450 mL). After confirming that the solution had a pH of 7.4±0.1, thedisulfide bond at the hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating thesolution at 22° C. for 10 minutes, a DMSO solution (0.559 mL; 9.2equivalents per antibody molecule) containing 10 mM of the compound inProcess 3 of Example 24 was added thereto and incubated for conjugatingthe drug linker to the antibody at 22° C. for 40 minutes. Next, anaqueous solution (0.112 mL; 18.4 equivalents per antibody molecule) of100 mM NAC was added thereto and incubated at 22° C. to terminate thereaction of the drug linker for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS6.0 was used as buffer solution) to yield asolution containing the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E, thefollowing characteristic values were obtained.

Antibody concentration: 10.65 mg/mL, antibody yield: 55.1 mg (61%), andaverage number of conjugated drug molecules (n) per antibody molecule:2.5.

Example 26 Antibody-Drug Conjugate (26)

Process 1: ({N-[(9H-Fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methylacetate

To a mixture containing N-9-fluorenylmethoxycarbonylglycylglycine (4.33g, 12.2 mmol), tetrahydrofuran (THF; 120 ml), and toluene (40.0 ml),pyridine (1.16 ml, 14.7 mmol) and lead tetraacetate (6.84 g, 14.7 mmol)were added and heated under reflux for 5 hours. After the reactionsolution was cooled to room temperature, the insolubles were removed byfiltration through Celite, and concentrated under reduced pressure. Theresidues obtained were dissolved in ethyl acetate and washed with waterand saturated brine, and then the organic layer was dried over anhydrousmagnesium sulfate. After the solvent was removed under reduced pressure,the residues obtained were purified by silica gel column chromatography[hexane: ethyl acetate=9:1 (v/v)-ethyl acetate] to yield the titledcompound as a colorless solid (3.00 g, 67%).

¹H-NMR (400 MHz, CDCl₃) δ: 2.07 (3H, s), 3.90 (2H, d, J=5.1 Hz), 4.23(1H, t, J=7.0 Hz), 4.46 (2H, d, J=6.6 Hz), 5.26 (2H, d, J=7.0 Hz), 5.32(1H, brs), 6.96 (1H, brs), 7.32 (2H, t, J=7.3 Hz), 7.41 (2H, t, J=7.3Hz), 7.59 (2H, d, J=7.3 Hz), 7.77 (2H, d, J=7.3 Hz).

Process 2:Benzyl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetate

To a THF (40.0 mL) solution of the compound (3.68 g, 10.0 mmol) obtainedin Process 1 above and benzyl glycolate (4.99 g, 30.0 mmol), potassiumtert-butoxide (2.24 g, 20.0 mmol) was added at 0° C. and stirred at roomtemperature for 15 minutes. The reaction solution was charged with ethylacetate and water at 0° C. and extracted with ethyl acetate andchloroform. The obtained organic layer was dried over sodium sulfate andfiltered. The solvent was removed under reduced pressure. The residuesobtained were dissolved in dioxane (40.0 mL) and water (10.0 mL),charged with sodium hydrogen carbonate (1.01 g, 12.0 mmol) and9-fluorenylmethyl chloroformate (2.59 g, 10.0 mmol), and stirred at roomtemperature for 2 hours. The reaction solution was charged with waterand extracted with ethyl acetate. The obtained organic layer was driedover sodium sulfate and filtered. The solvent was removed under reducedpressure and the residues obtained were purified by silica gel columnchromatography [hexane:ethyl acetate=100:0 (v/v)-0:100] to yield thetitled compound as a colorless oily substance (1.88 g, 40%).

¹H-NMR (400 MHz, CDCl₃) δ: 3.84 (2H, d, J=5.5 Hz), 4.24 (3H, t, J=6.5Hz), 4.49 (2H, d, J=6.7 Hz), 4.88 (2H, d, J=6.7 Hz), 5.15-5.27 (1H, m),5.19 (2H, s), 6.74 (1H, brs), 7.31-7.39 (7H, m), 7.43 (2H, t, J=7.4 Hz),7.61 (2H, d, J=7.4 Hz), 7.79 (2H, d, J=7.4 Hz).

Process 3:[({N-[(9H-Fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetic acid

The compound (1.88 g, 3.96 mmol) obtained in Process 2 above wasdissolved in ethanol (40.0 mL) and ethyl acetate (20.0 ml). After addingpalladium carbon catalyst (376 mg), it was stirred under hydrogenatmosphere at room temperature for 2 hours. The insolubles were removedby filtration through Celite, and the solvent was removed under reducedpressure to yield the titled compound as a colorless solid (1.52 g,quantitative).

¹H-NMR (400 MHz, DMSO-d₆) δ: 3.62 (2H, d, J=6.3 Hz), 3.97 (2H, s),4.18-4.32 (3H, m), 4.60 (2H, d, J=6.7 Hz), 7.29-7.46 (4H, m), 7.58 (1H,t, J=5.9 Hz), 7.72 (2H, d, J=7.4 Hz), 7.90 (2H, d, J=7.4 Hz), 8.71 (1H,t, J=6.5 Hz).

Process 4:9H-Fluoren-9-ylmethyl(2-{[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]amino}-2-oxoethyl)carbamate

Under ice cooling, to an N,N-dimethylformamide (10.0 mL) solution ofmethanesulfonic acid salt of exatecan (0.283 g, 0.533 mmol),N-hydroxysuccinimide (61.4 mg, 0.533 mmol), and the compound (0.205 g,0.533 mmol) obtained in Process 3 above, N,N-diisopropylethylamine (92.9μL, 0.533 mmol) and N,N′-dicyclohexylcarbodiimide (0.143 g, 0.693 mmol)were added and stirred at room temperature for 3 days. The solvent wasremoved under reduced pressure and the residues obtained were purifiedby silica gel column chromatography [chloroform-partitioned organiclayer of chloroform:methanol:water=7:3:1 (v/v/v)] to yield the titledcompound as a pale brown solid (0.352 g, 82%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.81 (3H, t, J=7.4 Hz), 1.73-1.87 (2H, m),2.06-2.20 (2H, m), 2.34 (3H, s), 3.01-3.23 (2H, m), 3.58 (2H, d, J=6.7Hz), 3.98 (2H, s), 4.13-4.25 (3H, m), 4.60 (2H, d, J=6.7 Hz), 5.09-5.22(2H, m), 5.32-5.42 (2H, m), 5.50-5.59 (1H, m), 6.49 (1H, s), 7.24-7.30(3H, m), 7.36 (2H, t, J=7.4 Hz), 7.53 (1H, t, J=6.3 Hz), 7.66 (2H, d,J=7.4 Hz), 7.75 (1H, d, J=11.0 Hz), 7.84 (2H, d, J=7.4 Hz), 8.47 (1H, d,J=8.6 Hz), 8.77 (1H, t, J=6.7 Hz).

MS (ESI) m/z: 802 (M+H)⁺

Process 5:N-[(2-{[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To an N,N-dimethylformamide (11.0 mL) solution of the compound (0.881 g,1.10 mmol) obtained in Process 4 above, piperidine (1.1 mL) was addedand stirred at room temperature for 2 hours. The solvent was removedunder reduced pressure to yield a mixture containing the titledcompound. The mixture was used for the next reaction without furtherpurification.

Process 6:N-[(9H-Fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

Under ice cooling, to an N,N-dimethylformamide (50.0 mL) solution of themixture (0.439 mmol) obtained in Process 5 above, N-hydroxysuccinimide(0.101 g, 0.878 mmol), andN-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanine (thecompound described in Japanese Patent Laid-Open No. 2002-60351; 0.440 g,0.878 mmol), N,N′-dicyclohexylcarbodiimide (0.181 g, 0.878 mmol) wasadded and stirred at room temperature for 4 days. The solvent wasremoved under reduced pressure and the residues obtained were purifiedby silica gel column chromatography [chloroform-chloroform:methanol=9:1(v/v)] to yield the titled compound as a pale orange solid (0.269 g,58%).

MS (ESI) m/z: 1063 (M+H)⁺

Process 7:Glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To an N,N-dimethylformamide (4.00 mL) solution of the compound (0.269 g,0.253 mmol) obtained in Process 6 above, piperidine (0.251 mL, 2.53mmol) was added and stirred at room temperature for 2 hours. The solventwas removed under reduced pressure to yield a mixture containing thetitled compound. The mixture was used for the next reaction withoutfurther purification.

Process 8:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To an N,N-dimethylformamide (10.0 mL) solution of the compound (0.253mmol) obtained in Process 7 above, N-succinimidyl 6-maleimide hexanoate(0.156 g, 0.506 mmol) was added and stirred at room temperature for 3days. The solvent was removed under reduced pressure and the residuesobtained were purified by silica gel column chromatography[chloroform-chloroform methanol=9:1 (v/v)] to yield the titled compoundas a pale yellow solid (0.100 g, 38%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.83 (3H, t, J=7.2 Hz), 1.09-1.21 (2H, m),1.33-1.47 (4H, m), 1.75-1.90 (2H, m), 2.00-2.23 (4H, m), 2.36 (3H, s),2.69-2.81 (1H, m), 2.94-3.03 (1H, m), 3.06-3.22 (2H, m), 3.23-3.74 (6H,m), 3.98 (2H, s), 4.39-4.50 (1H, m), 4.60 (2H, d, J=6.7 Hz), 5.17 (2H,s), 5.39 (2H, s), 5.53-5.61 (1H, m), 6.50 (1H, s), 6.96 (2H, s),7.11-7.24 (5H, m), 7.28 (1H, s), 7.75 (1H, d, J=11.0 Hz), 7.97 (1H, t,J=5.7 Hz), 8.03 (1H, t, J=5.9 Hz), 8.09 (1H, d, J=7.8 Hz), 8.27 (1H, t,J=6.5 Hz), 8.48 (1H, d, J=9.0 Hz), 8.60 (1H, t, J=6.5 Hz).

MS (ESI) m/z: 1034 (M+H)⁺

Process 9: Antibody-Drug Conjugate (26)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 8 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 2 of Example 6.

Antibody concentration: 1.61 mg/mL, antibody yield: 9.7 mg (77%), andaverage number of conjugated drug molecules (n) per antibody molecule:2.9.

Example 27 Antibody-Drug Conjugate (27)

Process 1: Antibody-Drug Conjugate (27)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 8 of Example 26, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 7.Antibody concentration: 1.58 mg/mL, antibody yield: 9.5 mg (76%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.6.

Example 28 Antibody-Drug Conjugate (28)

Process 1: Antibody-Drug Conjugate (28)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm⁻¹ was used). The solution (1.25 mL) was placed in two 1.5 mLpolypropylene tubes and charged with an aqueous solution of 10 mM TCEP(0.039 mL; 4.6 equivalents per antibody molecule) and an aqueoussolution of 1 M dipotassium hydrogen phosphate (0.0625 mL). Afterconfirming that the solution had a pH of 7.4±0.1, the disulfide bond atthe hinge part in the antibody was reduced by incubating at 37° C. for 1hour. Conjugation between antibody and drug linker: After adding DMSO(0.072 mL) and a DMSO solution containing 10 mM of the compound ofProcess 8 of Example 26 (0.078 mL; 9.2 equivalents per antibodymolecule) to the above solution at room temperature, it was stirred byusing a tube rotator for conjugating the drug linker to the antibody atroom temperature for 40 minutes. Next, an aqueous solution of 100 mM NAC(0.0155 mL) was added thereto and stirred at room temperature toterminate the reaction of the drug linker for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (ABS was used as a buffer solution) to yield 11.7mL in total of a solution containing the compound of interest.

Physicochemical characterization: By using the Common procedure E, thefollowing characteristic values were obtained.

Antibody concentration: 1.60 mg/mL, antibody yield: 18.7 mg (94%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.2.

Example 29 Antibody-Drug Conjugate (29)

Process 1: Antibody-Drug Conjugate (29)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm⁻¹ was used). The solution (6 mL) was placed in a polypropylene tubeand charged with an aqueous solution of 10 mM TCEP (0.108 mL; 2.5equivalents per antibody molecule) and an aqueous solution of 1 Mdipotassium hydrogen phosphate (0.091 mL). After confirming that thesolution had a pH of 7.0±0.1, the disulfide bond at the hinge part inthe antibody was reduced by incubating at 37° C. for 1 hour. Conjugationbetween antibody and drug linker: After adding DMSO (0.146 mL) and aDMSO solution containing 10 mM of the compound of Process 8 of Example26 (0.193 mL; 4.5 equivalents per antibody molecule) to the abovesolution at room temperature, it was incubated for conjugating the druglinker to the antibody at 15° C. for 1 hour. Next, an aqueous solution(0.029 mL) of 100 mM NAC was added thereto and stirred at roomtemperature to terminate the reaction of the drug linker for another 20minutes.

Purification: The above solution was subjected to purification using theCommon procedure D (ABS was used as buffer solution) to yield 24 mL of asolution containing the compound of interest.

Physicochemical characterization: By using the Common procedures E and F(ε_(D,280)=5178 (measured value), and ε_(D,370)=20217 (measured value)were used), the following characteristic values were obtained.

Antibody concentration: 1.77 mg/mL, antibody yield: 42 mg (85%), averagenumber of conjugated drug molecules (n) per antibody molecule measuredby the Common procedure E: 3.0, and average number of conjugated drugmolecules (n) per antibody molecule measured by the Common procedure F:3.4.

Example 30 Antibody-Drug Conjugate (30)

Process 1: Antibody-Drug Conjugate (30)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm⁻¹ was used). The solution (6 mL) was placed in a polypropylene tubeand charged with an aqueous solution of 10 mM TCEP (0.215 mL; 5equivalents per antibody molecule) and an aqueous solution of 1 Mdipotassium hydrogen phosphate (0.094 mL). After confirming that thesolution had a pH of 7.0±0.1, the disulfide bond at the hinge part inthe antibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After adding a DMSOsolution containing 10 mM of the compound of Process 8 of Example 26(0.370 mL; 8.6 equivalents per antibody molecule) to the above solutionat room temperature, it was incubated for conjugating the drug linker tothe antibody at 15° C. for 1 hour. Next, an aqueous solution (0.056 mL)of 100 mM NAC was added thereto and stirred at room temperature toterminate the reaction of the drug linker for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D (ABS was used as buffer solution) to yield 24 mL of asolution containing the compound of interest.

Physicochemical characterization: By using the Common procedures E and F(ε_(D,280)=5178 (measured value), and ε_(D,370)=20217 (measured value)were used), the following characteristic values were obtained.

Antibody concentration: 1.92 mg/mL, antibody yield: 46 mg (92%), averagenumber of conjugated drug molecules (n) per antibody molecule measuredby the Common procedure E: 6.2, and average number of conjugated drugmolecules (n) per antibody molecule measured by the Common procedure F:7.1.

Example 31 Antibody-Drug Conjugate (31)

Process 1: Antibody-Drug Conjugate (31)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm¹ was used). The solution (50.00 mL) was placed in a polypropylenecontainer and charged with an aqueous solution of 1 M dipotassiumhydrogen phosphate (0.745 mL) at room temperature with stirring and thenwith an aqueous solution of 10 mM TCEP (1.868 mL; 5.4 equivalents perantibody molecule). After confirming that the solution had a pH of7.0±0.1, stirring was terminated, and the disulfide bond at the hingepart in the antibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After cooling the abovesolution to 15° C., a DMSO solution containing 10 mM of the compound ofProcess 8 of Example 26 (2.958 mL; 8.6 equivalents per antibodymolecule) was gradually added dropwise thereto with stirring. While thetemperature was kept at 15° C., the reaction solution was stirred forthe first 30 minutes and incubated without stirring for conjugating thedrug linker to the antibody for the next 1 hour. Next, an aqueoussolution (0.444 mL) of 100 mM NAC was added thereto with stirring andstirred at room temperature to terminate the reaction of the drug linkerfor another 20 minutes.

Purification: By gradually adding 20% aqueous acetic acid solution(about 0.25 mL) and ABS (50 mL) to the above solution with stirring, thepH of the solution was adjusted to 5.5±0.1. This solution was subjectedto microfiltration (Millipore Corp., Millex-HV filter, 0.45 m, PVDFmembrane) to remove whitish matter. This solution was subjected toultrafiltration purification using an ultrafiltration apparatus composedof an ultrafiltration membrane (Merck Japan, Pellicon XL Cassette,Biomax 50 KDa), a tube pump (Cole-Parmer International, MasterFlex Pumpmodel 77521-40, Pump Head model 7518-00), and a tube (Cole-ParmerInternational, MasterFlex Tube L/S16). Specifically, while ABS was addeddropwise (a total of 800 mL) as a buffer solution for purification tothe reaction solution, ultrafiltration purification was performed forremoving unconjugated drug linkers and other low-molecular-weightreagents, also replacing the buffer solution with ABS, and furtherconcentrating the solution. The purified solution obtained was subjectedto microfiltration (0.22 m (Millipore Corp., Millex-GV filter, PVDFmembrane) and 0.10 m (Millipore Corp., Millex-VV filter, PVDF membrane))to yield a solution containing the titled antibody-drug conjugate.Physicochemical characterization: By using the Common procedures E and F(ε_(D,280)=5178 (measured value), and ε_(D,370)=20217 (measured value)were used), the following characteristic values were obtained.

Antibody concentration: 11.28 mg/mL, antibody yield: 451 mg (90%),average number of conjugated drug molecules (n) per antibody moleculemeasured by the Common procedure E: 6.6, and average number ofconjugated drug molecules (n) per antibody molecule measured by theCommon procedure F: 7.7.

Example 32 Alternative Method for Synthesizing Compound of Process 8 ofExample 26

Process 1: tert-ButylN-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalaninate

Under ice cooling, to a THF (12.0 ml) solution of tert-butylN-[(9H-fluoren-9-ylmethoxy) carbonyl]glycylglycyl-L-phenyl alaninate (J.Pept. Res., 1999, vol. 53, pp. 393; 0.400 g, 0.717 mmol),1,8-diazabicyclo[5.4.0]-7-undecene (0.400 ml) was added and stirred atroom temperature for 4 days, and then N-succinimidyl 6-maleimidehexanoate (0.221 g, 0.717 mmol) was further added and stirred for 3hours. The reaction solution was diluted with ethyl acetate and washedwith an aqueous solution of 10% citric acid, a saturated aqueoussolution of sodium hydrogen carbonate, and saturated brine, and then theorganic layer was dried over anhydrous magnesium sulfate. After thesolvent was removed under reduced pressure, the residues obtained werepurified by silica gel column chromatography [chloroform-chloroformmethanol=9:1 (v/v)] to yield the titled compound as a pale yellow solid(0.295 g, 78%).

¹H-NMR (400 MHz, CDCl₃) δ: 1.28-1.36 (2H, m), 1.41 (9H, s), 1.57-1.71(4H, m), 2.23 (2H, t, J=7.6 Hz), 3.09 (2H, d, J=6.0 Hz), 3.51 (2H, t,J=7.6 Hz), 3.85-4.02 (4H, m), 4.69-4.78 (1H, m), 6.15 (1H, t, J=4.6 Hz),6.33 (1H, d, J=7.3 Hz), 6.60 (1H, t, J=5.0 Hz), 6.68 (2H, s), 7.10-7.16(2H, m), 7.22-7.31 (3H, m).

MS (ESI) m/z: 529 (M+H)⁺

Process 2:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanine

To a dichloromethane (8.00 ml) solution of the compound (0.295 g, 0.558mmol) obtained in Process 1 above, trifluoroacetic acid (4.00 mL) wasadded and stirred at room temperature for 18 hours. The solvent wasremoved under reduced pressure to yield the titled compound as a paleyellow solid (0.240 g, 91%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.15-1.23 (2H, m), 1.40-1.53 (4H, m), 2.10(2H, t, J=7.6 Hz), 2.88 (1H, dd, J=13.7, 8.9 Hz), 3.04 (1H, dd, J=13.7,5.0 Hz), 3.35-3.43 (2H, m), 3.58-3.77 (4H, m), 4.41 (1H, td, J=7.8, 5.0Hz), 7.00 (2H, s), 7.16-7.31 (5H, m), 8.00 (1H, t, J=5.7 Hz), 8.06 (1H,t, J=5.7 Hz), 8.13 (1H, d, J=7.8 Hz).

Process 3:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

The compound (0.572 g, 1.21 mmol) obtained in Process 2 above wasdissolved in dichloromethane (12.0 mL), charged withN-hydroxysuccinimide (0.152 g, 1.32 mmol) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.253 g,1.32 mmol), and stirred for 1 hour. The reaction solution was added toan N,N-dimethylformamide (22.0 mL) solution of the mixture (1.10 mmol)obtained in Process 5 of Example 26, and stirred at room temperature for3 hours. The reaction solution was charged with an aqueous solution of10% citric acid and extracted with chloroform. The obtained organiclayer was dried over sodium sulfate and filtered. The solvent wasremoved under reduced pressure and the residues obtained were purifiedby silica gel column chromatography [chloroform-chloroform:methanol=8:2(v/v)] to yield the titled compound as a pale yellow solid (0.351 g,31%). The instrumental data of the compound was the same as that of thecompound of Process 8 of Example 26.

Example 33 Alternative Method for Synthesizing Compound of Process 8 ofExample 26

Process 1:Benzyl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetate

To a THF (200 ml) solution of the compound (7.37 g, 20.0 mmol) obtainedin Process 1 of Example 26, benzyl glycolate (6.65 g, 40.0 mmol) andp-toluene sulfonic acid monohydrate (0.381 g, 2.00 mmol) were added at0° C. and stirred at room temperature for 2 hours and 30 minutes. Thereaction solution was charged with a saturated aqueous solution ofsodium hydrogen carbonate and extracted with ethyl acetate. The obtainedorganic layer was dried over sodium sulfate and filtered. The solventwas removed under reduced pressure and the residues obtained werepurified by silica gel column chromatography [hexane: ethylacetate=100:0 (v/v)-0:100] to yield the titled compound as a colorlesssolid (6.75 g, 71%). The instrumental data of the compound was the sameas that of the compound of Process 2 of Example 26.

Process 2:N-[(Benzyloxy)carbonyl]glycylglycyl-L-phenylalanine-N-{[(2-(benzyloxy)-2-oxoethoxy]methyl}glycinamide

To an N,N-dimethylformamide (140 mL) solution of the compound (6.60 g,13.9 mmol) obtained in Process 1 above,1,8-diazabicyclo[5.4.0]undec-7-ene (2.22 g, 14.6 mmol) was added at 0°C. and stirred at room temperature for 15 minutes. The reaction solutionwas charged with an N,N-dimethylformamide (140 mL) solution ofN-[(benzyloxy)carbonyl]glycylglycyl-L-phenylalanine (6.33 g, 15.3 mmol),N-hydroxysuccinimide (1.92 g, 16.7 mmol), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.20 g,16.7 mmol) stirred in advance at room temperature for 1 hour, andstirred at room temperature for 4 hours. The reaction solution wascharged with 0.1 N hydrochloric acid and extracted with chloroform. Theobtained organic layer was dried over sodium sulfate and filtered. Thesolvent was removed under reduced pressure and the residues obtainedwere purified by silica gel column chromatography[chloroform-chloroform:methanol=8:2 (v/v)] to yield the titled compoundas a colorless solid (7.10 g, 79%).

¹H-NMR (DMSO-d₆) δ: 2.78 (1H, dd, J=13.9, 9.6 Hz), 3.05 (1H, dd, J=13.9,4.5 Hz), 3.56-3.80 (6H, m), 4.15 (2H, s), 4.47-4.55 (1H, m), 4.63 (2H,d, J=6.6 Hz), 5.03 (2H, s), 5.15 (2H, s), 7.16-7.38 (15H, m), 7.52 (1H,t, J=5.9 Hz), 8.03 (1H, t, J=5.5 Hz), 8.17 (1H, d, J=8.2 Hz), 8.36 (1H,t, J=5.7 Hz), 8.61 (1H, t, J=6.6 Hz).

Process 3:Glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide

To an N,N-dimethylformamide (216 mL) solution of the compound (7.00 g,10.8 mmol) obtained in Process 2 above, palladium carbon catalyst (7.00g) was added and stirred under a hydrogen atmosphere at room temperaturefor 24 hours. The insolubles were removed by filtration through Celite,and the solvent was removed under reduced pressure. The residuesobtained were dissolved in water, the insoluble material was removed byfiltration through Celite, and the solvent was removed under reducedpressure. This procedure was repeated twice to yield the titled compoundas a colorless solid (3.77 g, 82%).

¹H-NMR (DMSO-d₆) δ: 2.84 (1H, dd, J=13.7, 9.8 Hz), 3.08 (1H, dd, J=13.7,4.7 Hz), 3.50-3.72 (4H, m), 3.77-3.86 (2H, m), 3.87 (2H, s), 4.52-4.43(1H, m), 4.61 (2H, d, J=6.6 Hz), 7.12-7.30 (5H, m), 8.43 (1H, t, J=5.9Hz), 8.54 (1H, d, J=7.8 Hz), 8.70 (1H, t, J=6.3 Hz), 8.79 (1H, t, J=5.5Hz).

Process 4:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide

To an N,N-dimethylformamide (85.0 mL) solution of the compound (3.59 g,8.48 mmol) obtained in Process 3 above, N-succinimidyl 6-maleimidehexanoate (2.88 g, 9.33 mmol) and triethylamine (0.858 g, 8.48 mmol)were added and stirred at room temperature for 1 hour. The reactionsolution was charged with 0.1 N hydrochloric acid and extracted withchloroform and a mixed solvent of chloroform and methanol[chloroform:methanol=4:1 (v/v)]. The obtained organic layer was driedover sodium sulfate and filtered. The solvent was removed under reducedpressure and the residues obtained were purified by silica gel columnchromatography [chloroform-partitioned organic layer ofchloroform:methanol:water=7:3:1 (v/v/v)] to yield the titled compound asa colorless solid (3.70 g, 71%).

¹H-NMR (DMSO-d₆) δ: 1.13-1.24 (2H, m), 1.42-1.53 (4H, m), 2.11 (2H, t,J=7.4 Hz), 2.80 (1H, dd, J=13.7, 9.8 Hz), 3.06 (1H, dd, J=13.9, 4.5 Hz),3.37 (2H, t, J=7.2 Hz), 3.56-3.78 (6H, m), 3.97 (2H, s), 4.46-4.53 (1H,m), 4.61 (2H, d, J=6.3 Hz), 7.00 (2H, s), 7.15-7.29 (5H, m), 8.03-8.20(3H, m), 8.32 (1H, t, J=5.9 Hz), 8.60 (1H, t, J=6.7 Hz).

Process 5:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To an N,N-dimethylformamide (40.0 mL) solution of methanesulfonic acidsalt of exatecan (1.14 g, 2.00 mmol), triethylamine (0.202 g, 2.00mmol), the compound (1.48 g, 2.40 mmol) obtained in Process 4 above, and4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(0.993 g, 3.00 mmol) containing 16.4% water were added at 0° C. andstirred at room temperature for 1 hour. The solvent was removed underreduced pressure and the residues obtained were purified by silica gelcolumn chromatography [chloroform-chloroform methanol=8:2 (v/v)] toyield the titled compound as a pale yellow solid (1.69 g, 82%). Thespectral data of the compound was the same as that of the compound ofProcess 8 of Example 26.

Example 34 Intermediate (34)

Process1:2-{[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethylacetate

Under ice cooling, to an N,N-dimethylformamide (20.0 mL) suspension ofmethanesulfonic acid salt of exatecan (0.500 g, 0.941 mmol),N,N-diisopropylethylamine (0.492 mL, 2.82 mmol) and acetoxyacetylchloride (0.121 ml, 1.13 mmol) were added and stirred at roomtemperature for 1 hour. The solvent was removed under reduced pressureand the residues obtained were purified by silica gel columnchromatography [chloroform-partitioned organic layer ofchloroform:methanol:water=7:3:1 (v/v/v)] to yield the titled compound asa pale yellow solid (0.505 g, quantitative).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.81-1.92 (2H, m),2.08 (3H, s), 2.08-2.22 (2H, m), 2.41 (3H, s), 3.14-3.21 (2H, m), 4.51(2H, dd, J=19.4, 14.7 Hz), 5.22 (2H, dd, J=40.1, 19.0 Hz), 5.43 (2H, s),5.56-5.61 (1H, m), 6.53 (1H, s), 7.31 (1H, s), 7.81 (1H, d, J=11.0 Hz),8.67 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 536 (M+H)⁺

Process 2:N-[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamide

To a methanol (50.0 mL) suspension of the compound (0.504 g, 0.941 mmol)obtained in Process 1 above, a THF (20.0 ml) and an aqueous solution of1 N sodium hydroxide (4.00 ml, 4.00 mmol) were added and stirred at roomtemperature for 1 hour. The reaction was terminated by the addition of 1N hydrochloric acid (5.00 ml, 5.00 mmol), and the solvent was removedunder reduced pressure. The residues obtained were purified by silicagel column chromatography [chloroform-partitioned organic layer ofchloroform:methanol water=7:3:1 (v/v/v)] to yield the titled compound asa pale yellow solid (0.412 g, 89%). This compound was confirmed in thetumor of a mouse that received the antibody-drug conjugate (45) or (46).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.3 Hz), 1.78-1.95 (2H, m),2.09-2.28 (2H, m), 2.39 (3H, s), 3.07-3.27 (2H, m), 3.96 (2H, d, J=6.0Hz), 5.11-5.26 (2H, m), 5.42 (2H, s), 5.46-5.54 (1H, m), 5.55-5.63 (1H,m), 6.52 (1H, s), 7.30 (1H, s), 7.78 (1H, d, J=10.9 Hz), 8.41 (1H, d,J=9.1 Hz). MS (ESI) m/z: 494 (M+H)⁺

Example 35 Alternative Method for Synthesizing Compound of Example 34

Process 1:N-[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamideGlycolic acid (0.0201 g, 0.27 mmol) was dissolved inN,N-dimethylformamide (1.0 mL), charged with N-hydroxysuccinimide(0.0302 g, 0.27 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (0.0508 g, 0.27 mmol), and stirred for 1 hour. Thereaction solution was added to an N,N-dimethylformamide suspension (1.0mL) charged with methanesulfonic acid salt of exatecan (0.1 g, 0.176mmol) and triethylamine (0.025 mL, 0.18 mmol) and stirred at roomtemperature for 24 hours. The solvent was removed under reduced pressureand the residues obtained were purified by silica gel columnchromatography [chloroform-chloroform:methanol=10:1 (v/v)] to yield thetitled compound as a pale yellow solid (0.080 g, 92%). The spectral dataof the compound was the same as that of the compound obtained in Process2 of Example 34.

Example 36 Antibody-Drug Conjugate (36)

Process 1:N-[4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[,2-b]quinolin-1-yl]-β-alaninamide

The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 15was reacted in the same manner as Process 3 of Example 2 by usingN-succinimidyl 4-maleimide butyrate instead of N-succinimidyl6-maleimide hexanoate to yield the titled compound as a pale white solid(24.0 mg, 38%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.2 Hz), 1.68 (2H, quin,J=7.4 Hz), 1.78-1.92 (2H, m), 2.06-2.22 (2H, m), 2.10 (2H, t, J=7.8 Hz),2.31-2.43 (2H, m), 2.40 (3H, s), 2.78 (1H, dd, J=13.7, 9.4 Hz), 3.01(1H, dd, J=13.7, 4.7 Hz), 3.17 (4H, d, J=5.1 Hz), 3.29-3.40 (2H, m),3.52-3.80 (6H, m), 4.40-4.51 (1H, m), 5.19 (1H, d, J=18.4 Hz), 5.26 (1H,d, J=18.8 Hz), 5.42 (2H, s), 5.52-5.61 (1H, m), 6.53 (1H, s), 6.99 (2H,s), 7.12-7.28 (5H, m), 7.31 (1H, s), 7.74-7.84 (2H, m), 8.02 (1H, t,J=5.9 Hz), 8.08-8.16 (2H, m), 8.25 (1H, t, J=5.9 Hz), 8.52 (1H, d, J=8.2Hz).

MS (ESI) m/z: 990 (M+H)⁺

Process 2: Antibody-Drug Conjugate (33)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 2 of Example 6.

Antibody concentration: 1.75 mg/mL, antibody yield: 10.5 mg (84%), andaverage number of conjugated drug molecules (n) per antibody molecule:4.7.

Example 37 Antibody-Drug Conjugate (37)

Process 1: Antibody-Drug Conjugate (37)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1 of Example 36, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 7.

Antibody concentration: 1.89 mg/mL, antibody yield: 11.3 mg (90%), andaverage number of conjugated drug molecules (n) per antibody molecule:8.5.

Example 38 Intermediate (38)

Process 1: tert-Butyl(5-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-5-oxopentyl)carbamate

Methanesulfonic acid salt of exatecan (500 mg, 0.941 mmol) was reactedin the same manner as Process 1 of Example 1 by using5-(tert-butoxycarbonylamino)valeric acid instead of4-(tert-butoxycarbonylamino)butanoic acid to yield the titled compoundas a yellow-brown solid (571 mg, 96%). The compound was used for thenext reaction without performing further purification.

MS(ESI)m/z:635(M+H)⁺

Process 2:5-Amino-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]pentanamide

The compound (558 mg, 0.879 mmol) obtained in Process 1 above wasreacted in the same manner as Process 2 of Example 1 to yieldtrifluoroacetate of the titled compound as a yellow solid (363 mg, 64%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.88 (3H, t, J=7.4 Hz), 1.52-1.71 (4H, m),1.87 (2H, tt, J=14.4, 6.9 Hz), 2.07-2.18 (2H, m), 2.22 (2H, t, J=7.0Hz), 2.40 (3H, s), 2.76-2.88 (2H, m), 3.13-3.22 (2H, m), 5.18 (1H, d,J=18.8 Hz), 5.24 (1H, d, J=18.8 Hz), 5.43 (2H, s), 5.53-5.61 (1H, m),6.55 (1H, s), 7.33 (1H, s), 7.65 (3H, br.s.), 7.81 (1H, d, J=11.3 Hz),8.49 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 535 (M+H)⁺

Example 39 Antibody-Drug Conjugate (39)

Process 1:N-(tert-Butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-(5-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-5-oxopentyl)glycinamide

The compound (348 mg, 0.537 mmol) obtained in Process 2 of Example 38was reacted in the same manner as Process 1 of Example 2 to yield thetitled compound as a pale yellow solid (429 mg, 84%). The compound wasused for the next reaction without performing further purification.

Process 2:Glycylglycyl-L-phenylalanyl-N-(5-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-5-oxopentyl)glycinamide

The compound (427 mg, 0.448 mmol) obtained in Process 1 above wasreacted in the same manner as Process 2 of Example 2 to yieldtrifluoroacetate of the titled compound as a yellow solid (430 mg, 99%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.2 Hz), 1.38-1.49 (2H, m),1.54-1.66 (2H, m), 1.86 (2H, tt, J=14.5, 7.0 Hz), 2.08-2.16 (2H, m),2.19 (2H, t, J=7.2 Hz), 2.40 (3H, s), 2.76 (1H, dd, J=13.9, 10.0 Hz),3.00-3.12 (3H, m), 3.14-3.21 (2H, m), 3.57 (2H, d, J=4.7 Hz), 3.60-3.75(3H, m), 3.87 (1H, dd, J=16.8, 5.9 Hz), 4.55 (1H, td, J=9.0, 4.7 Hz),5.16 (1H, d, J=18.8 Hz), 5.23 (1H, d, J=18.4 Hz), 5.44 (2H, s),5.53-5.60 (1H, m), 6.55 (1H, s), 7.14-7.29 (5H, m), 7.32 (1H, s), 7.74(1H, t, J=5.5 Hz), 7.81 (1H, d, J=10.9 Hz), 7.96 (3H, br.s.), 8.30-8.37(1H, m), 8.44-8.53 (2H, m).

MS (ESI) m/z: 853 (M+H)⁺

Process 3:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(5-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-5-oxopentyl)glycinamide

The compound (60.0 mg, 0.0621 mmol) obtained in Process 2 above wasreacted in the same manner as Process 3 of Example 2 to yield the titledcompound as a solid (16.0 mg, 25%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.13-1.21 (2H, m),1.36-1.52 (6H, m), 1.53-1.65 (2H, m), 1.79-1.92 (2H, m), 2.05-2.15 (4H,m), 2.19 (2H, s), 2.40 (3H, s), 2.79 (1H, dd, J=13.7, 10.2 Hz),2.98-3.10 (3H, m), 3.12-3.21 (2H, m), 3.29-3.37 (2H, m), 3.53-3.79 (6H,m), 4.41-4.50 (1H, m), 5.16 (1H, d, J=18.8 Hz), 5.23 (1H, d, J=18.8 Hz),5.43 (2H, s), 5.52-5.60 (1H, m), 6.53 (1H, s), 6.99 (2H, s), 7.12-7.28(5H, m), 7.31 (1H, s), 7.63 (1H, t, J=5.7 Hz), 7.80 (1H, d, J=10.6 Hz),8.02 (1H, t, J=5.9 Hz), 8.08 (1H, t, J=5.7 Hz), 8.12 (1H, d, J=7.8 Hz),8.24 (1H, t, J=5.7 Hz), 8.45 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 1046 (M+H)⁺

Process 4: Antibody-Drug Conjugate (39)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure C-1 and Common procedure B (asabsorption coefficient at 280 nm, 1.37 mLmg⁻¹ cm⁻¹ was used). Thesolution (1.0 mL) was collected into a 2 mL tube and charged with anaqueous solution of 10 mM TCEP (0.0155 mL; 2.3 equivalents per antibodymolecule) and an aqueous solution of 1 M dipotassium hydrogen phosphate(0.050 mL). After confirming that the solution had a pH of 7.4±0.1, thedisulfide bond at the hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating thesolution for 10 minutes at 22° C., a DMSO solution (0.0311 mL; 4.6equivalents per antibody molecule) containing 10 mM of the compoundobtained in Process 3 was added thereto and incubated for conjugatingthe drug linker to the antibody at 22° C. for 40 minutes. Next, anaqueous solution (0.00622 mL; 9.2 equivalents per antibody molecule) of100 mM NAC was added thereto and incubated at 22° C. to terminate thereaction of the drug linker for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS6.0 was used as buffer solution) to yield 6 mLof a solution containing the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E, thefollowing characteristic values were obtained.

Antibody concentration: 1.12 mg/mL, antibody yield: 6.72 mg (67%), andaverage number of conjugated drug molecules (n) per antibody molecule:1.8.

Example 40 Antibody-Drug Conjugate (40)

Process 1: Antibody-Drug Conjugate (40)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL withPBS6.0/EDTA by using the Common procedure C-1 and Common procedure B (asabsorption coefficient at 280 nm, 1.37 mLmg⁻¹ cm⁻¹ was used). Thesolution (1.0 mL) was collected into a 2 mL tube and charged with anaqueous solution of 10 mM TCEP (0.0311 mL; 4.6 equivalents per antibodymolecule) and an aqueous solution of 1 M dipotassium hydrogen phosphate(0.050 mL). After confirming that the solution had a pH of 7.4±0.1, thedisulfide bond at the hinge part in the antibody was reduced byincubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After incubating thesolution at 22° C. for 10 minutes, a DMSO solution (0.0622 mL; 9.2equivalents per antibody molecule) containing 10 mM of the compoundobtained in Process 3 of Example 39 was added thereto and incubated forconjugating the drug linker to the antibody at 22° C. for 40 minutes.Next, an aqueous solution (0.0124 mL; 18.4 equivalents per antibodymolecule) of 100 mM NAC was added thereto and incubated at 22° C. toterminate the reaction of the drug linker for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D-1 (PBS6.0 was used as buffer solution) to yield 6 mLof a solution containing the titled antibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E, thefollowing characteristic values were obtained.

Antibody concentration: 0.98 mg/mL, antibody yield: 5.88 mg (59%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.4.

Example 41 Antibody-Drug Conjugate (41)

Process 1: tert-Buty {2-[(2-hydroxyethyl)amino]-2-oxoethyl}carbamate

N-(tert-Butoxycarbonyl)glycine (4.2 g, 24 mmol) was dissolved indimethylformamide (40 mL). After adding aminoethanol (2.9 g, 48 mmol)and 1-hydroxybenzotriazole (3.7 g, 24 mmol) and adding1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (6.9 g, 36mmol), it was stirred at room temperature for 12 hours. The solvent wasremoved under reduced pressure, and the residue was charged with toluenefor azeotropic distillation. The residue obtained were purified bysilica gel column chromatography [ethyl acetate-ethyl acetatemethanol=10:1 (v/v)] to yield the titled compound as a colorless oilysubstance (3.8 g, 72%).

¹H-NMR (400 MHz, CDCl₃) δ: 1.44 (9H, s), 1.69 (1H, brs), 3.43 (2H, td,J=5.9, 5.1 Hz), 3.71 (2H, t, J=5.1 Hz), 3.79 (2H, d, J=5.9 Hz), 5.22(1H, brs), 6.62 (1H, brs).

Process 2:2-{[N-(tert-Butoxycarbonyl)glycyl]amino}ethyl4-nitrophenylcarbonate

To a THF (23 mL) solution of the compound (1.0 g, 4.59 mmol) obtained inProcess 1 above, diisopropylethylamine (0.80 mL, 4.59 mmol) andbis(4-nitrophenyl) carbonate (1.32 g, 6.88 mmol) were added and stirredat room temperature for 12 hours. The solvent was removed under reducedpressure, and the residue obtained was purified by silica gel columnchromatography [hexane-hexane: ethyl acetate=1:3 (v/v)] to yield thetitled compound as a pale yellow solid (1.13 g, 64%).

¹H-NMR (400 MHz, CDCl₃) δ: 1.44 (1H, s), 3.66 (2H, td, J=5.1, 5.9 Hz),3.81 (2H, d, J=5.9 Hz), 4.36 (2H, t, J=5.1 Hz), 5.07 (1H, s), 6.48-6.53(1H, m), 7.38 (2H, dt, J=9.9, 2.7 Hz), 8.27 (2H, dt, J=9.9, 2.7 Hz).

Process 3:2-({[(tert-Butoxycarbonyl)amino]acetyl}amino)ethyl[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]carbamate

To methanesulfonic acid salt of exatecan (0.70 g, 1.2 mmol), thecompound (0.57 g, 1.5 mmol) obtained in Process 2, and1-hydroxybenzotriazole (3.7 g, 24 mmol), dimethylformamide (23 mL) wasadded, and diisopropylethylamine (0.43 mL, 2.5 mmol) was added andstirred at room temperature for 12 hours. The solvent was removed underreduced pressure, and the residue was charged with toluene forazeotropic distillation. The residue obtained were purified by silicagel column chromatography [chloroform-chloroform:methanol=10:1 (v/v)] toyield the titled compound (0.86 g, quantitative) as a pale yellow solid.

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.35 (9H, s),1.78-1.94 (1H, m), 2.07-2.17 (1H, m), 2.17-2.27 (1H, m), 2.37 (3H, s),3.05-3.16 (1H, m), 3.19-3.26 (1H, m), 3.34-3.39 (2H, m), 3.50-3.56 (2H,m), 4.00-4.07 (1H, m), 4.13-4.21 (1H, m), 5.15-5.34 (3H, m), 5.44 (2H,s), 6.54 (1H, s), 6.90-6.96 (1H, m), 7.32 (1H, s), 7.78 (1H, d, J=11.0Hz), 7.93-8.07 (2H, m).

Process 4:2-(Glycylamino)ethyl[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]carbamate

The compound (0.86 g, 2.1 mmol) obtained in Process 3 above wasdissolved in dichloromethane (15 mL). After adding trifluoroacetic acid(15 mL), it was stirred for 1 hour. The solvent was removed underreduced pressure, and the residue was charged with toluene forazeotropic distillation. The residue obtained were purified by silicagel column chromatography [chloroform-partitioned organic layer ofchloroform:methanol:water=7:3:1 (v/v/v)] to yield the titled compound asa pale yellow solid (0.86 g, 99%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.2 Hz), 1.79-1.95 (2H, m),2.06-2.18 (1H, m), 2.18-2.29 (1H, m), 2.38 (3H, s), 3.07-3.17 (1H, m),3.20-3.29 (1H, m), 3.36-3.50 (2H, m), 3.51-3.62 (2H, m), 3.99-4.08 (1H,m), 4.22-4.31 (1H, m), 5.16-5.35 (3H, m), 5.42 (1H, d, J=18.8 Hz), 5.46(1H, d, J=18.8 Hz), 6.56 (1H, s), 7.34 (1H, s), 7.65 (2H, brs), 7.79(1H, d, J=10.6 Hz), 7.99-8.06 (1H, m), 8.51 (1H, t, J=5.5 Hz).

MS (APCI) m/z: 939 (M+H)⁺

Process 5:N-[(9H-Fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanyl-N-[2-({[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]carbamoyl}oxy)ethyl]glycinamide

N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanine(Japanese Patent Laid-Open No. 2002-60351; 0.21 g, 0.41 mmol) wasdissolved in N,N-dimethylformamide (3 mL). After addingN-hydroxysuccinimide (0.052 g, 0.45 mmol) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.086 g,0.45 mmol), it was stirred for 1 hour. The reaction solution was addeddropwise to a N,N-dimethylformamide solution (2 mL) charged with thecompound (0.24 g, 0.35 mmol) obtained in Process 4 and triethylamine(0.078 mL, 0.45 mmol), and stirred at room temperature for 1 hour. Thesolvent was removed under reduced pressure, and the residue obtained waspurified by silica gel column chromatography[chloroform-chloroform:methanol=8:2 (v/v)] to yield the titled compoundas a pale yellow solid (0.24 g, 65%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.3 Hz), 1.79-1.90 (2H, m),2.05-2.27 (2H, m), 2.36 (3H, s), 2.73-2.81 (1H, m), 2.98-3.12 (2H, m),3.17-3.26 (1H, m), 3.35-3.42 (2H, m), 3.55-3.79 (6H, m), 4.00-4.10 (1H,m), 4.12-4.23 (2H, m), 4.23-4.29 (2H, m), 4.45-4.55 (1H, m), 5.13-5.33(3H, m), 5.40 (1H, d, J=17.2 Hz), 5.44 (1H, d, J=17.2 Hz), 6.53 (1H, s),7.11-7.26 (5H, m), 7.26-7.33 (3H, m), 7.38 (2H, t, J=7.6 Hz), 7.57 (1H,t, J=5.9 Hz), 7.68 (2H, d, J=7.4 Hz), 7.77 (1H, d, J=11.0 Hz), 7.85 (2H,d, J=9.0 Hz), 7.91-7.97 (1H, m), 7.98-8.05 (2H, m), 8.14 (1H, d, J=7.8Hz), 8.31-8.26 (1H, m).

MS (APCI) m/z: 1063 (M+H)⁺

Process 6:Glycylglycyl-L-phenylalanyl-N-[2-({[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]carbamoyl}oxy)ethyl]glycinamide

The compound (0.24 g, 0.35 mmol) obtained in Process 5 above was reactedin the same manner as Process 7 of Example 26 to yield the titledcompound as a pale yellow solid (0.12 g, 65%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.4 Hz), 1.78-1.94 (2H, m),2.06-2.27 (2H, m), 2.37 (3H, s), 2.72-2.81 (1H, m), 2.98-3.07 (1H, m),3.12-3.17 (2H, m), 3.57-3.81 (6H, m), 4.00-4.21 (3H, m), 4.45-4.54 (1H,m), 5.15-5.35 (3H, m), 5.41 (1H, d, J=17.2 Hz), 5.45 (1H, d, J=17.2 Hz),6.54 (1H, s), 7.11-7.26 (6H, m), 7.32 (1H, s), 7.78 (1H, d, J=11.0 Hz),7.93-8.00 (1H, m), 8.03 (1H, d, J=9.4 Hz), 8.06-8.13 (1H, m), 8.21-8.27(2H, m), 8.30-8.36 (1H, m).

MS (APCI) m/z: 841 (M+H)⁺

Process 7:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[2-({[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]carbamoyl}oxy)ethyl]glycinamide

The compound (42.0 mg, 0.0499 mmol) obtained in Process 6 was reacted inthe same manner as Process 3 of Example 2 to yield the titled compoundas a pale yellow solid (38.3 mg, 74%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.87 (3H, t, J=7.4 Hz), 1.12-1.23 (2H, m),1.40-1.51 (4H, m), 1.80-1.95 (2H, m), 2.05-2.27 (4H, m), 2.38 (3H, s),3.43-2.40 (8H, m), 3.53-3.78 (6H, m), 4.00-4.21 (2H, m), 4.44-4.55 (1H,m), 5.17-5.36 (3H, m), 5.43 (2H, s), 6.54 (1H, s), 6.99 (2H, s), 7.19(5H, d, J=23.9 Hz), 7.33 (1H, s), 7.78 (1H, d, J=10.6 Hz), 7.91-8.16(5H, m), 8.24-8.31 (1H, m).

MS (ESI) m/z: 1034 (M+H)⁺

Process 8: Antibody-Drug Conjugate (41)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 7 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 2 of Example 6.

Antibody concentration: 1.54 mg/mL, antibody yield: 9.2 mg (74%), andaverage number of conjugated drug molecules (n) per antibody molecule:3.7.

Example 42 Antibody-Drug Conjugate (42)

Process 1: Antibody-Drug Conjugate (42)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 7 of Example 41, the titled antibody-drugconjugate was obtained in the same manner as Process 1 of Example 7.Antibody concentration: 1.47 mg/mL, antibody yield: 8.8 mg (71%), andaverage number of conjugated drug molecules (n) per antibody molecule:7.0.

Example 43 Antibody-Drug Conjugate (43)

Process 1:N-{3-[2-(2-{[3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]propanoyl}glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

The compound (53.7 mg, 50.5 mol) obtained in Process 6 of Example 26 wasdissolved in N,N-dimethylformamide (1.50 mL). After adding1,8-diazabicyclo(5.4.0)-7-undecene (7.5 μL, 50.5 μmol), it was stirredat room temperature for 30 minutes. The reaction solution was chargedwith pyridinium p-toluenesulfonate (14.0 mg, 5.56 mol), then chargedwith N-succinimidyl3-(2-(2-(3-maleinimidopropanamido)ethoxy)ethoxy)propanoate (32.3 mg,75.8 mol), and stirred at room temperature for 2.25 hours. The solventwas removed under reduced pressure, and the residue obtained waspurified by silica gel column chromatography [[chloroform-partitionedorganic layer of chloroform:methanol water=7:3:1 (v/v/v)] to yield thetitled compound as a pale yellow solid (27.1 mg, 47%).

¹H-NMR (DMSO-d₆) δ: 0.87 (3H, t, J=7.0 Hz), 1.79-1.91 (2H, m), 2.18 (2H,t, J=15.1 Hz), 2.29-2.33 (4H, m), 2.39 (3H, s), 2.76 (1H, dd, J=13.9,9.2 Hz), 3.02 (1H, dd, J=13.7, 3.9 Hz), 3.13-3.15 (2H, m), 3.44-3.46(6H, m), 3.57-3.59 (6H, m), 3.69-3.75 (6H, m), 4.01 (2H, s), 4.46-4.48(1H, m), 4.63 (2H, d, J=6.3 Hz), 5.21 (2H, s), 5.42 (2H, s), 5.60 (1H,dd, J=13.5, 5.7 Hz), 6.54 (1H, s), 7.00 (2H, s), 7.17-7.24 (6H, m), 7.31(1H, s), 7.79 (1H, d, J=11.0 Hz), 8.00-8.02 (2H, m), 8.13 (1H, d, J=7.8Hz), 8.17 (1H, t, J=6.3 Hz), 8.52 (1H, d, J=9.0 Hz), 8.65 (1H, t, J=6.5Hz).

MS (ESI) m/z=1151 (M+H)⁺

Process 2: Antibody-Drug Conjugate (43)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 1, the titled antibody-drug conjugate wasobtained in the same manner as Process 1 of Example 7.

Antibody concentration: 1.96 mg/mL, antibody yield: 17.6 mg (88%), andaverage number of conjugated drug molecules (n) per antibody molecule:5.6.

Example 44 Antibody-Drug Conjugate (44)

Process 1: tert-ButylN-[(benzyloxy)carbonyl]glycylglycyl-D-phenylalaninate

N-[(Benzyloxy)carbonyl]glycylglycine (3.00 g, 11.3 mmol) was dissolvedin N,N-dimethylformamide (20.0 mL). After adding N-hydroxysuccinimide(1.43 g, 12.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (2.37 g, 12.4 mmol), it was stirred for 1 hour. AN,N-dimethylformamide solution (10 mL) charged with D-phenylalaninetert-butyl (2.74 g, 12.38 mmol) and triethylamine (1.73 mL, 12.4 mmol)was added dropwise to the reaction solution and stirred at roomtemperature for 2 hours. The reaction solution was charged withdichloromethane and washed with water, 1 N hydrochloric acid, and asaturated aqueous solution of sodium bicarbonate, and then the organiclayer was dried over anhydrous sodium sulfate. The solvent was removedunder reduced pressure, and the residue obtained was purified by silicagel column chromatography [chloroform-chloroform:methanol=9:1 (v/v)] toyield the titled compound as a colorless solid (4.21 g, 80%).

¹H-NMR (CDCl₃) δ: 1.41 (9H, s), 3.03-3.14 (2H, m), 3.86-3.97 (4H, m),4.70-4.77 (1H, m), 5.13 (2H, s), 5.43 (1H, brs), 6.42 (1H, d, J=10.0Hz), 6.64-6.71 (1H, m), 7.11-7.15 (2H, m), 7.20-7.31 (4H, m), 7.31-7.38(4H, m).

MS (APCI) m/z: 470 (M+H)⁺

Process 2: N-[(Benzyloxy)carbonyl]glycylglycyl-D-phenylalanine

The compound (4.21 g, 8.97 mmol) obtained in Process 1 was dissolved inethyl acetate (20 mL). After adding an ethyl acetate solution (20.0 mL)of 4 N hydrogen chloride, it was left overnight at room temperature. Thesolvent was removed under reduced pressure, then toluene was added, andthe solvent was removed under reduced pressure. The residue obtained waspurified by silica gel column chromatography [chloroform-partitionedorganic layer of chloroform: methanol:water=7:3:1 (v/v/v)] to yield thetitled compound as a colorless solid (1.66 g, 45%).

¹H-NMR (CDCl₃) δ: 2.92-3.01 (1H, m), 3.10-3.18 (1H, m), 3.65-3.81 (3H,m), 3.88-3.98 (1H, m), 4.64-4.73 (1H, m), 5.06 (2H, s), 5.87 (1H, brs),7.10-7.37 (13H, m).

MS (APCI) m/z: 412 (M+H)⁻

Process 3:N-[(Benzyloxy)carbonyl]glycylglycyl-D-phenylalanyl-N-{[2-(benzyloxy)-2-oxoethoxy]methyl}glycinamide

To a dioxane (25.0 mL) solution of the compound (1.25 g, 2.63 mmol)obtained in Process 1 of Example 32, piperidine (5.00 mL) andN,N-dimethylformamide (5.00 mL) were added and stirred at roomtemperature for 30 minutes. The solvent was removed under reducedpressure, and the residue obtained was dissolved inN,N-dimethylformamide (20.0 mL). After adding the compound (1.20 g, 2.90mmol) of Process 2 above and4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.03g, 3.16 mmol) containing 16.4% water, it was stirred at room temperaturefor 2 hours. The reaction solution was charged with chloroform andwashed with water, and then the organic layer was dried over anhydroussodium sulfate. The solvent was removed under reduced pressure, and theresidue obtained was purified by silica gel column chromatography[chloroform-chloroform:methanol=9:1 (v/v)] to yield the titled compoundas a colorless solid (270 mg, 16%).

¹H-NMR (DMSO-d₆) δ: 2.78 (1H, dd, J=13.6, 10.0 Hz), 3.05 (1H, dd,J=13.9, 4.2 Hz), 3.56-3.79 (6H, m), 4.15 (2H, s), 4.47-4.54 (1H, m),4.63 (2H, d, J=6.7 Hz), 5.03 (2H, s), 5.15 (2H, s), 7.14-7.39 (15H, m),7.50 (1H, t, J=5.7 Hz), 8.02 (1H, t, J=5.4 Hz), 8.16 (1H, d, J=7.9 Hz),8.34 (1H, t, J=6.0 Hz), 8.60 (1H, t, J=7.0 Hz).

MS (APCI) m/z: 648 (M+H)⁺

Process 4:Glycylglycyl-D-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide

The compound (200 mg, 0.31 mmol) obtained in Process 3 above wasdissolved in N,N-dimethylformamide (5.0 mL). After adding 5% palladiumcarbon catalyst (0.12 g), it was stirred under a hydrogen atmosphere atroom temperature for 9 hours. The reaction solution was filtered throughCelite, and the residue was washed with a mixed solvent of water andN,N-dimethylformamide. The filtrate and the wash were combined, and thesolvent was removed under reduced pressure to yield the titled compoundas a colorless solid (0.15 g, quantitative).

¹H-NMR (DMSO-d₆) δ: 2.85 (1H, dd, J=13.3, 9.7 Hz), 3.08 (1H, dd, J=13.9,5.4 Hz), 3.43-3.52 (4H, m), 3.62-3.89 (7H, m), 4.36-4.44 (1H, m),4.58-4.67 (2H, m), 7.12-7.29 (5H, m), 8.44 (1H, t, J=5.7 Hz), 8.67 (1H,d, J=7.3 Hz), 8.78 (1H, t, J=5.4 Hz), 8.91 (1H, brs).

MS (APCI) m/z: 424 (M+H)⁺

Process 5:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-D-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide

The compound (0.15 g, 0.35 mmol) obtained in Process 4 above wasdissolved in N,N-dimethylformamide (10 mL). After adding N-succinimidyl6-maleimide hexanoate (0.11 g, 0.35 mmol), it was stirred at roomtemperature for 1 hour. The reaction solution was charged withchloroform and washed with water, and then the organic layer was driedover anhydrous sodium sulfate. The solvent was removed under reducedpressure, and the residue obtained was purified by silica gel columnchromatography [chloroform-partitioned organic layer ofchloroform:methanol water=7:3:1 (v/v/v)] to yield the titled compound asa colorless solid (41 mg, 26%).

¹H-NMR (DMSO-d₆) δ: 1.13-1.24 (2H, m), 1.42-1.53 (4H, m), 2.12 (2H, t,J=7.3 Hz), 2.82 (1H, dd, J=13.9, 10.0 Hz), 3.09 (1H, dd, J=13.9, 4.8Hz), 3.17 (2H, d, J=4.2 Hz), 3.47-3.89 (8H, m), 4.08-4.14 (1H, m),4.41-4.49 (1H, m), 4.58-4.69 (2H, m), 7.00 (2H, s), 7.14-7.27 (5H, m),8.31 (1H, t, J=6.0 Hz), 8.39 (1H, brs), 8.55 (2H, brs), 8.93 (1H, brs).

MS (APCI) m/z: 615 (M−H)⁻

Process 6:N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-D-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To a N,N-dimethylformamide (10 mL) solution of methanesulfonic acid saltof exatecan (22 mg, 0.388 mmol), triethylamine (5.42 μL, 0.388 mmol),the compound (29 mg, 0.466 mmol) obtained in Process 5 above, and4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (19mg, 0.686 mmol) containing 16.4% water were added at 0° C. and stirredat room temperature for 1 hour. The solvent in the reaction solution wasremoved under reduced pressure, and then the residue obtained waspurified by silica gel column chromatography [chloroform-partitionedorganic layer of chloroform:methanol:water=7: 3:1 (v/v/v)] to yield thetitled compound as a pale yellow solid (26 mg, 65%).

¹H-NMR (DMSO-d₆) δ: 0.87 (3H, t, J=7.3 Hz), 1.12-1.22 (2H, m), 1.40-1.51(4H, m), 1.79-1.92 (2H, m), 2.09 (2H, t, J=7.6 Hz), 2.13-2.23 (2H, m),2.39 (3H, s), 2.78 (1H, dd, J=13.6, 9.4 Hz), 2.98-3.05 (1H, m),3.13-3.23 (2H, m), 3.54-3.78 (8H, m), 4.02 (2H, s), 4.41-4.50 (1H, m),4.61-4.66 (2H, m), 5.21 (2H, s), 5.42 (2H, s), 5.56-5.64 (1H, m), 6.53(1H, s), 6.99 (2H, s), 7.14-7.27 (5H, m), 7.31 (1H, s), 7.79 (1H, d,J=10.9 Hz), 8.01 (1H, t, J=5.4 Hz), 8.07 (1H, t, J=5.7 Hz), 8.14 (1H, d,J=7.9 Hz), 8.31 (1H, t, J=5.7 Hz), 8.53 (1H, d, J=9.1 Hz), 8.63 (1H, t,J=6.3 Hz).

MS (APCI) m/z: 1034 (M+H)⁺

Process 7: Antibody-Drug Conjugate (44)

By using the trastuzumab produced in Reference Example 1 and thecompound obtained in Process 6 above, the titled antibody-drug conjugatewas obtained in the same manner as Process 1 of Example 7.

Antibody concentration: 1.87 mg/mL, antibody yield: 16.8 mg (84%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.1.

Example 45 Intermediate (45)

Process 1: tert-Butyl(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethyl)carbamate

To a dichloromethane (3.00 mL) solution ofN-(tert-butoxycarbonyl)-glycine (0.395 g, 2.26 mmol),N-hydroxysuccinimide (0.260 g, 2.26 mmol) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.433 mg,2.26 mmol) were added and stirred at room temperature for 1 hour. Thissolution was added to a solution consisting of methanesulfonic acid saltof exatecan (1.00 g, 1.88 mmol), triethylamine (0.315 mL, 2.26 mmol),and N,N-dimethylformamide (3.00 mL) and stirred at room temperature for16.5 hours. The reaction solution was diluted with chloroform and washedwith 10% citric acid solution, and then the organic layer was dried overanhydrous sodium sulfate. The solvent was removed under reducedpressure, and the residue obtained was purified by silica gel columnchromatography [chloroform-chloroform:methanol=9:1 (v/v)] to yield thetitled compound as a yellow solid (1.16 g, 99%).

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.86 (3H, t, J=7.2 Hz), 1.30 (9H, s),1.81-1.89 (2H, m), 2.09-2.21 (2H, m), 2.38 (3H, s), 3.15-3.17 (2H, m),3.55-3.56 (2H, m), 5.15 (1H, d, J=18.8 Hz), 5.23 (1H, d, J=19.2 Hz),5.41 (2H, s), 5.55-5.56 (1H, m), 6.53 (1H, s), 6.95 (1H, t, J=5.5 Hz),7.28 (1H, s), 7.77 (1H, d, J=11.0 Hz), 8.39 (1H, d, J=8.6 Hz).

MS (APCI) m/z: 593 (M+H)⁺

Process 2:N-[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]glycinamide

The compound (0.513 g, 1.01 mmol) obtained in Process 1 above wasreacted in the same manner as Process 2 of Example 1 to yield the titledcompound as a yellow solid (0.463 g, 93%).

¹H-NMR (400 MHz, CD₃OD) δ: 0.96 (3H, t, J=7.0 Hz), 1.89-1.91 (2H, m),2.14-2.16 (1H, m), 2.30 (3H, s), 2.40-2.42 (1H, m), 3.15-3.21 (2H, m),3.79-3.86 (2H, m), 4.63-4.67 (1H, m), 5.00-5.05 (1H, m), 5.23 (1H, d,J=16.0 Hz), 5.48 (1H, d, J=16.0 Hz), 5.62-5.64 (1H, m), 7.40-7.45 (2H,m).

MS (APCI) m/z: 493 (M+H)⁺

Example 46 Antibody-Drug Conjugate (46)

Process 1: Antibody-Drug Conjugate (46)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm⁻¹ was used). The solution (50 mL) was placed in a 125 mLpolycarbonate Erlenmeyer flask, charged with an aqueous solution of 1 Mdipotassium hydrogen phosphate at room temperature (0.750 mL) withstirring using a magnetic stirrer, and then charged with an aqueoussolution of 10 mM TCEP (1.857 mL; 5.4 equivalents per antibodymolecule). After confirming that the solution had a pH of 7.0±0.1,stirring was terminated, and the disulfide bond at the hinge part in theantibody was reduced by incubating at 37° C. for 1 hour.

Conjugation between antibody and drug linker: After cooling the abovesolution to 15° C., a DMSO solution containing 10 mM of the compound ofProcess 8 of Example 26 (2.958 mL; 8.6 equivalents per antibodymolecule) was gradually added dropwise thereto with stirring. At 15° C.,the reaction solution was stirred for the first 30 minutes and incubatedwithout stirring for conjugating the drug linker to the antibody for thenext 1 hour. Next, an aqueous solution (0.444 mL; 12.9 equivalents perantibody molecule) of 100 mM NAC was added thereto with stirring andstirred at room temperature to terminate the reactivity of unreacteddrug linkers for another 20 minutes.

Purification: By gradually adding 20% aqueous acetic acid solution(about 0.25 mL) and ABS (50 mL) to the above solution with stirring, thepH of the solution was adjusted to 5.5±0.1. This solution was subjectedto microfiltration (Millipore Corp., Millex-HV filter, 0.45 m, PVDFmembrane) to remove whitish matter. This solution was subjected toultrafiltration purification using an ultrafiltration apparatus composedof an ultrafiltration membrane (Merck Japan, Pellicon XL Cassette,Biomax 50 KDa), a tube pump (Cole-Parmer International, MasterFlex Pumpmodel 77521-40, Pump Head model 7518-00), and a tube (Cole-ParmerInternational, MasterFlex Tube L/S16). Specifically, while ABS was addeddropwise (a total of 800 mL) as a buffer solution for purification tothe reaction solution, ultrafiltration purification was performed forremoving unconjugated drug linkers and other low-molecular-weightreagents, also replacing the buffer solution with ABS, and furtherconcentrating the solution. The purified solution obtained was subjectedto microfiltration (0.22 μm (Millipore Corp., Millex-GV filter, PVDFmembrane) and 0.10 μm (Millipore Corp., Millex-VV filter, PVDFmembrane)) to yield 42.5 mL of a solution containing the titledantibody-drug conjugate. Physicochemical characterization: By using theCommon procedures E and F (ε_(D,280)=5178 (measured value), andε_(D,370)=20217 (measured value) were used), the followingcharacteristic values were obtained.

Antibody concentration: 10.4 mg/mL, antibody yield: 442 mg (88.5%),average number of conjugated drug molecules (n) per antibody moleculemeasured by the Common procedure E: 6.0, and average number ofconjugated drug molecules (n) per antibody molecule measured by theCommon procedure F: 7.5.

Example 47 Antibody-Drug Conjugate (47)

Process 1: Antibody-Drug Conjugate (47)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm¹ was used). The solution (15 mL) was placed in a polypropylene tubeand charged with an aqueous solution of 10 mM TCEP (0.567 mL; 5.5equivalents per antibody molecule) and an aqueous solution of 1 Mdipotassium hydrogen phosphate (0.225 mL). After confirming that thesolution had a pH of 7.0±0.1, the disulfide bond at the hinge part inthe antibody was reduced by incubating at 37° C. for 2 hour. Conjugationbetween antibody and drug linker: After adding DMSO (0.146 mL) and aDMSO solution containing 10 mM of the compound of Process 8 of Example26 (0.928 mL; 9.0 equivalents per antibody molecule) to the abovesolution at room temperature, it was incubated for conjugating the druglinker to the antibody at 15° C. for 30 minutes. Next, an aqueoussolution (0.133 mL; 12.9 equivalents per antibody molecule) of 100 mMNAC was added thereto and stirred at room temperature to terminate thereactivity of unreacted drug linkers for another 20 minutes.

Purification: The above solution was subjected to purification using theCommon procedure D (ABS was used as buffer solution) to yield 49 mL of asolution containing the compound of interest.

Physicochemical characterization: By using the Common procedure E(ε_(D,280)=5178 and ε_(D,370)=20217 were used), the followingcharacteristic values were obtained.

Antibody concentration: 2.91 mg/mL, antibody yield: 143 mg (95%), andaverage number of conjugated drug molecules (n) per antibody molecule:6.2.

Example 48 Antibody-Drug Conjugate (48)

Process 1: Antibody-Drug Conjugate (48)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm¹ was used). The solution (280 mL) was placed in a 1000 mLpolycarbonate Erlenmeyer flask, charged with an aqueous solution of 1 Mdipotassium hydrogen phosphate at room temperature (4.200 mL) withstirring using a magnetic stirrer, and then charged with an aqueoussolution of 10 mM TCEP (10.594 mL; 5.5 equivalents per antibodymolecule). After confirming that the solution had a pH of 7.4±0.1,stirring was terminated, and the disulfide bond at the hinge part in theantibody was reduced by incubating at 37° C. for 2 hour.

Conjugation between antibody and drug linker: After cooling the abovesolution to 15° C., a DMSO solution containing 10 mM of the compound ofProcess 8 of Example 26 (17.335 mL; 9.0 equivalents per antibodymolecule) was gradually added dropwise thereto with stirring. Thereaction solution was stirred at 15° C. for conjugating the drug linkerto the antibody for 30 minutes. Next, an aqueous solution (2.485 mL;12.9 equivalents per antibody molecule) of 100 mM NAC was added theretowith stirring and stirred at room temperature to terminate thereactivity of unreacted drug linkers for another 20 minutes.

Purification: By gradually adding 20% aqueous acetic acid solution(about 1.4 mL) and ABS (280 mL) to the above solution with stirring, thepH of the solution was adjusted to 5.5±0.1. This solution was subjectedto microfiltration (0.45 m, PVDF membrane) to remove whitish matterwhile yielding about 600 mL of a filtrate. This solution was subjectedto ultrafiltration purification using an ultrafiltration apparatuscomposed of an ultrafiltration membrane (Merck Japan, Pellicon XLCassette, Biomax 50 KDa), a tube pump (Cole-Parmer International,MasterFlex Pump model 77521-40, Pump Head model 7518-00), and a tube(Cole-Parmer International, MasterFlex Tube L/S16). Specifically, whileABS was added dropwise (a total of 4800 mL) as a buffer solution forpurification to the reaction solution, ultrafiltration purification wasperformed for removing unconjugated drug linkers and otherlow-molecular-weight reagents, also replacing the buffer solution withABS, and further concentrating the solution. The purified solutionobtained was subjected to microfiltration (twice with 0.22 μm and 0.10μm, PVDF membrane) to yield 70 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E(ε_(D,280)=5178 and ε_(D,370)=20217 were used), the followingcharacteristic values were obtained. Antibody concentration: 35.96mg/mL, antibody yield: 2517 mg (90%), and average number of conjugateddrug molecules (n) per antibody molecule: 6.2.

Example 49 Antibody-Drug Conjugate (49)

Process 1: Antibody-Drug Conjugate (49)

Reduction of the antibody: The trastuzumab produced in Reference Example1 was prepared to have an antibody concentration of 10 mg/mL byreplacing the medium with PBS6.0/EDTA by using the Common procedure C-1and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg⁻¹cm⁻¹ was used). The solution (280 mL) was placed in a 1000 mLpolycarbonate Erlenmeyer flask, charged with an aqueous solution of 1 Mdipotassium hydrogen phosphate at room temperature (4.200 mL) withstirring using a magnetic stirrer, and then charged with an aqueoussolution of 10 mM TCEP (10.594 mL; 5.5 equivalents per antibodymolecule). After confirming that the solution had a pH of 7.0±0.1,stirring was terminated, and the disulfide bond at the hinge part in theantibody was reduced by incubating at 37° C. for 2 hour.

Conjugation between antibody and drug linker: After cooling the abovesolution to 15° C., a DMSO solution containing 10 mM of the compound ofProcess 8 of Example 26 (17.335 mL; 9.0 equivalents per antibodymolecule) was gradually added dropwise thereto with stirring. Thereaction solution was stirred at 15° C. for conjugating the drug linkerto the antibody for 30 minutes. Next, an aqueous solution (2.485 mL;12.9 equivalents per antibody molecule) of 100 mM NAC was added theretowith stirring and stirred at room temperature to terminate thereactivity of unreacted drug linkers for another 20 minutes.

Purification: By gradually adding 20% aqueous acetic acid solution(about 1.4 mL) and ABS (280 mL) to the above solution with stirring, thepH of the solution was adjusted to 5.5±0.1. This solution was subjectedto microfiltration (0.45 m, PVDF membrane) to remove whitish matterwhile yielding about 600 mL of a filtrate. This solution was subjectedto ultrafiltration purification using an ultrafiltration apparatuscomposed of an ultrafiltration membrane (Merck Japan, Pellicon XLCassette, Ultracell 30 KDa), a tube pump (Cole-Parmer International,MasterFlex Pump model 77521-40, Pump Head model 7518-00), and a tube(Cole-Parmer International, MasterFlex Tube L/S16). Specifically, whileABS was added dropwise (a total of 4800 mL) as a buffer solution forpurification to the reaction solution, ultrafiltration purification wasperformed for removing unconjugated drug linkers and otherlow-molecular-weight reagents, also replacing the buffer solution withABS, and further concentrating the solution. The purified solutionobtained was subjected to microfiltration (twice with 0.22 m and 0.10 m,PVDF membrane) to yield 130 mL of a solution containing the titledantibody-drug conjugate.

Physicochemical characterization: By using the Common procedure E(ε_(D,280)=5178 and ε_(D,370)=20217 were used), the followingcharacteristic values were obtained. Antibody concentration: 21.00mg/mL, antibody yield: 2730 mg (97.5%), and average number of conjugateddrug molecules (n) per antibody molecule: 6.3.

Example 50 Antibody-Drug Conjugate (50) Process 1: Antibody-DrugConjugate (50)

The antibody-drug conjugates (47), (48), and (49) produced in Examples47, 48, and 49 were mixed (243 mL) and further charged with ABS (39.75mL) to yield 283 mL of a solution containing the titled antibody-drugconjugate.

Physicochemical characterization: By using the Common procedures E and F(ε_(D,280=5178), and ε_(D,370)=20217 were used), the followingcharacteristic values were obtained.

Antibody concentration: 20.0 mg/mL, antibody yield: 5655 mg, averagenumber of conjugated drug molecules (n) per antibody molecule measuredby the Common procedure E: 6.3, and average number of conjugated drugmolecules (n) per antibody molecule measured by the Common procedure F:7.8.

Evaluation Example 1 Cell Growth Inhibitory Effect (1) of Antibody-DrugConjugate

Human breast cancer line KPL-4 of HER2 antigen-positive cells (Dr.Junichi Kurebayashi, Kawasaki Medical School, British Journal of Cancer,(1999) 79 (5/6). 707-717) or human breast cancer line MCF7 ofantigen-negative cells (European Collection of Cell Cultures; ECACC) wascultured in RPMI1640 (GIBCO) containing 10% fetal bovine serum(MOREGATE) (hereinafter, referred to as medium). The KPL-4 or MCF7 wasprepared to have a concentration of 2.5×10⁴ cells/mL by using medium,added at a concentration of 100 μL/well to a 96-well microplate for cellculture, and cultured overnight.

On the next day, trastuzumab or the antibody-drug conjugate diluted into1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and 0.064 nM by usingmedium was added at a concentration of 10 μL/well to the microplate.Medium was added at a concentration of 10 μL/well to antibodynon-supplemented wells. The cells were cultured under 5% CO₂ at 37° C.for 5 to 7 days. After the culture, the microplate was taken out fromthe incubator and left standing at room temperature for 30 minutes. Theculture solution was charged with an equal amount of CellTiter-GloLuminescent Cell Viability Assay (Promega) and stirred. After themicroplate was left standing at room temperature for 10 minutes,luminescence intensity of each well was measured by using a plate reader(PerkinElmer). The IC₅₀ value was calculated according to the followingequation:

IC ₅₀ (nM)=antilog((50−d)×(LOG₁₀(b)−LOG₁₀(a))/(d−c)+LOG₁₀(b))

a: Concentration of sample ab: Concentration of sample bc: Cell viability of sample ad: Cell viability of sample b

The cell viability at each concentration was calculated according to thefollowing equation:

Cell viability (%)=a/b×100

a: Average luminescence intensity of the sample wells (n=2)b: Average luminescence intensity of the antibody non-supplemented wells(n=10)

The antibody-drug conjugates (2), (3), (5), (7), (10), (12), (13), (16),(18), (40), and (42) exhibited a cell growth inhibitory effect ofIC₅₀<0.1 (nM) against the KPL-4 cells.

The antibody-drug conjugates (4), (6), (9), (15), (17), (21), (22),(25), (36), (37), (39), (41), and (43) exhibited a cell growthinhibitory effect of 0.1<IC₅₀<1 (nM) against the cells.

The antibody-drug conjugates (20), (24), and (27) exhibited a cellgrowth inhibitory effect of 1<IC₅₀<100 (nM) against the cells. Neitherof the antibody-drug conjugates (19) or (26) exhibited a cell growthinhibitory effect against the cells (>100 (nM)).

On the other hand, the antibody-drug conjugates (5), (13), and (43)exhibited a cell growth inhibitory effect of 1<IC₅₀<100 (nM) against theMCF7 cells, whereas none of the antibody-drug conjugates (2), (3), (4),(6), (7), (9), (10), (12), (15), (16), (17), (18), (25), (26), (27),(39), (40), (41), (42), and (44) exhibited a cell growth inhibitoryeffect against the cells (>100 (nM)).

Trastuzumab exhibited a cell growth inhibitory effect against neitherthe KPL-4 cells nor the MCF7 cells (>100 (nM)).

Evaluation Example 2 Antitumor Test (1)

Mouse: 5- to 6-week-old female nude mice (Charles River LaboratoriesJapan, Inc.) were acclimatized for 4 to 7 days under SPF conditionsbefore use in the experiment. The mice were fed with sterilized solidfeed (FR-2, Funabashi Farms Co., Ltd) and given sterilized tap water(prepared by the addition of 5 to 15 ppm sodium hypochlorite solution).

Assay and calculation expression: In all studies, the major axis andminor axis of a tumor were measured twice a week by using an electronicdigital caliper (CD-15CX, Mitutoyo Corp.), and the tumor volume (mm³)was calculated. The calculation expression is as shown below.

Tumor volume (mm³)=1/2×Major axis (mm)×[Minor axis (mm)]²

All of the antibody-drug conjugates and the antibody were diluted withphysiological saline (Otsuka Pharmaceutical Factory, Inc.) and used at avolume of 10 mL/kg for intravenous administration to the tail vein ofeach mouse.

KPL-4 cells were suspended in physiological saline, and 1.5×10⁷ cellswere subcutaneously transplanted to the right side of the body of eachfemale nude mouse (Day 0), and the mice were randomly grouped on Day 15.The antibody-drug conjugate (27) or the anti-HER2 antibody trastuzumab(Reference Example 1) for a control group was intravenously administeredat a dose of 10 mg/kg to the tail vein of each mouse at Days 15 and 22.An untreated group was established as a control group.

The results are shown in FIG. 3. The administration of trastuzumabinhibited tumor growth, whereas the administration of the antibody-drugconjugate (27) produced a more significant tumor growth inhibitoryeffect. In the drawing, the abscissa depicts days after tumorinoculation, and the ordinate depicts tumor volume. In addition, themice that received trastuzumab or the antibody-drug conjugate (27) werefree from notable signs such as weight loss, suggesting that theantibody-drug conjugate (27) is highly safe. In the Evaluation Examplesbelow regarding the antitumor test, the test was conducted by theprocedure used in this Evaluation Example, unless otherwise specified.

Evaluation Example 3 Antitumor Test (2)

Human gastric cancer line NCI-N87 cells purchased from ATCC (AmericanType Culture Collection) were suspended in physiological saline, and1×10⁷ cells were subcutaneously transplanted to the right side of thebody of each female nude mouse (Day 0), and the mice were randomlygrouped on Day 7. The antibody-drug conjugate (8) or (28), ortrastuzumab emtansine (Reference Example 2) was intravenouslyadministered at a dose of 10 mg/kg to the tail vein of each mouse on Day7. An untreated group was established as a control group.

The results are shown in FIG. 4. The antibody-drug conjugates (8) and(28) were confirmed to have a strong antitumor effect with tumorregression equivalent to that of trastuzumab emtansine. In addition, theadministration of the antibody-drug conjugate (8) or (28), ortrastuzumab emtansine was found to be free from weight loss of the mice.

Evaluation Example 4 Antitumor Test (3)

Human breast cancer line JIMT-1 cells purchased from DSMZ (DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH) were suspended inphysiological saline, and 3×10⁶ cells were subcutaneously transplantedto the right side of the body of each female nude mouse (Day 0), and themice were randomly grouped on Day 12. The antibody-drug conjugate (8),(29), or (30), trastuzumab, or trastuzumab emtansine was intravenouslyadministered at a dose of 10 mg/kg to the tail vein of each mouse onDays 12 and 19. A physiological saline administration group wasestablished as a control group.

The results are shown in FIG. 5. The administration of trastuzumab ortrastuzumab emtansine did not inhibit the growth of the JIMT-1 tumor. Onthe other hand, the administration of the antibody-drug conjugate (8),(29), or (30) inhibited the growth of the tumor. In addition, theadministration of the antibody-drug conjugate (8), (29), or (30),trastuzumab, or trastuzumab emtansine was found to be free from weightloss of the mice.

Evaluation Example 5 Cell Growth Inhibitory Effect (2) of Antibody-DrugConjugate

Human non-small cell lung cancer line Calu-3 (ATCC) was cultured inEagle's Minimum Essential Medium (GIBCO) containing 10% fetal bovineserum (MOREGATE) (hereinafter, referred to as MEM medium).

Human gastric cancer line NCI-N87 (ATCC) or human gastric cancer lineMKN-45 (Health Science Research Resources Bank) was cultured in RPMI1640Medium (GIBCO) containing 10% fetal bovine serum (hereinafter, referredto as RPMI medium).

Human breast cancer line MDA-MB-453 (ATCC) or human breast cancer lineMDA-MB-468 (ATCC) was cultured in Leibovitz's L-15 Medium (GIBCO)containing 10% fetal bovine serum (hereinafter, referred to asLeibovitz's medium).

Among these 5 types of cell lines, Calu-3, NCI-N87, and MDA-MB-453 areHER2-positive cells, and MKN-45 and MDA-MB-468 are HER2-negative cells.

Calu-3, NCI-N87, or MKN-45 was prepared to have a concentration of 4×10⁴cells/mL by using MEM medium or RPMI medium, added at a concentration of25 μlL/well to a 96-well microplate for cell culture charged with 65μL/well of a medium, and cultured overnight under 5% CO₂ at 37° C. Also,MDA-MB-453 or MDA-MB-468 was prepared to have a concentration of 4×10⁴cells/mL by using Leibovitz's medium, added at a concentration of 25μL/well to a 96-well microplate for cell culture charged with 65 μL/wellof medium, and cultured overnight at 37° C. without setting CO₂concentration.

On the next day, a test substance diluted into 1000 nM, 200 nM, 40 nM, 8nM, 1.6 nM, 0.32 nM, and 0.064 nM by using RPMI medium or Leibovitz'smedium, or RPMI medium or Leibovitz's medium was added at aconcentration of 10 L/well to the microplate. The cells were culturedunder 5% CO₂ at 37° C. or at 37° C. without setting CO₂ concentrationfor 6 days.

For Calu-3, NCI-N87, and MDA-MB-468, the antibody-drug conjugate (46)was added as the test substance. For the other cells, the antibody-drugconjugate (50) was added as the test substance. After the culture, themicroplate was taken out from the incubator and left standing at roomtemperature for 30 minutes. The culture solution was charged with anequal amount of CellTiter-Glo Luminescent Cell Viability Assay (Promega)and stirred with a plate mixer to completely lyse the cells. After themicroplate was left standing at room temperature for 10 minutes,luminescence intensity was measured by using a plate reader.

Cell viability was calculated according to the following equation:

Cell viability (%)=a/b×100

a: Average luminescence intensity of the test substance-supplementedwellsb: Average luminescence intensity of the medium-supplemented wells

The IC₅₀ value was calculated according to the following equation:

IC ₅₀ (nM)=antilog((50−d)×(LOG₁₀(b)−LOG₁₀(a))/(d−c)+LOG₁₀(b))

a: Concentration a of the test substanceb: Concentration b of the test substancec: Cell viability supplemented with the test substance having theconcentration ad: Cell viability supplemented with the test substance having theconcentration b

The concentrations a and b establish the relation a>b crossing 50% ofcell viability.

The antibody-drug conjugate (46) exhibited a cell growth inhibitoryeffect of IC₅₀<1 (nM) against the HER2-positive cells Calu-3 andNCI-N87. On the other hand, the antibody-drug conjugate (46) exhibitedno cell growth inhibitory effect against the HER2-negative cellsMDA-MB-468 (>100 (nM)).

The antibody-drug conjugate (50) exhibited a cell growth inhibitoryeffect of IC₅₀<1 (nM) against the HER2-positive cells MDA-MB-453. On theother hand, the antibody-drug conjugate (50) exhibited no cell growthinhibitory effect against the HER2-negative cells MKN-45 (>100 (nM)).

Evaluation Example 6 Antitumor Test (4)

Human pancreatic cancer line Capan-1 cells (ATCC) weakly expressing HER2were suspended in physiological saline, and 4×10⁷ cells weresubcutaneously transplanted to the right side of the body of each femalenude mouse to generate Capan-1 solid tumor. Thereafter, this solid tumorwas maintained at several passages by transplantation to female nudemice and used in this test. A tumor section of the solid tumor wassubcutaneously transplanted to the right side of the body of each femalenude mouse (Day 0), and the mice were randomly grouped on Day 20.

The antibody-drug conjugate (31), trastuzumab, or trastuzumab emtansinewas intravenously administered at a dose of 10 mg/kg to the tail vein ofeach mouse on Day 20. A physiological saline administration group wasestablished as a control group.

The results are shown in FIG. 6. The administration of trastuzumab ortrastuzumab emtansine did not inhibit the growth of the Capan-1 tumor.By contrast, the administration of the antibody-drug conjugate (31)inhibited the growth of the tumor, demonstrating that the antibody-drugconjugate (31) is effective for even tumor with low HER2 expression. Theantibody-drug conjugate (31) did not inhibit the growth of HER2non-expressing gastric cancer line GCIY tumor.

As for the expression of HER2 in tumor, on the basis of measurementresults of immunohistochemical staining described in the 3rd edition ofthe guidelines for HER2 testing (developed by the Japanese PathologyBoard for Optimal Use of Trastuzumab, The Japanese Society ofPathology), classification was performed such that score of 3+: highexpression, 2+: moderate expression, and 1+: low expression. Even if thescore was 0 in this measurement method, tumor found HER2-positive byother measurement methods such as a measurement method using a flowcytometer was classified as low expressing tumor.

Evaluation Example 7 Antitumor Test (5)

Human gastric cancer line NCI-N87 cells purchased from ATCC weresuspended in physiological saline, and 1×10⁷ cells were subcutaneouslytransplanted to the right side of the body of each female nude mouse(Day 0), and the mice were randomly grouped on Day 6. The antibody-drugconjugate (50) was intravenously administered at each dose of 0.3, 1, 3,or 10 mg/kg to the tail vein of each mouse on Day 6. An acetate buffersolution administration group was established as a control group.

The results are shown in FIG. 7. The antibody-drug conjugate (50)exhibited a dose-dependent antitumor effect. In addition, theadministration of the antibody-drug conjugate (50) was found to be freefrom weight loss of the mice.

Evaluation Example 8 Antitumor Test (6)

This test was carried out by the following method.

Mouse: 6- to 12-week-old female nude mice (Charles River LaboratoriesJapan, Inc.) were subjected to the experiment.

Assay and calculation expression: The major axis and minor axis of tumorwere measured twice a week by using an electronic digital caliper, andthe tumor volume (mm³) was calculated. The calculation expression is asshown below.

Tumor volume (mm³)=0.52×Major axis(mm)×[Minor axis (mm)]²

The antibody-drug conjugate, trastuzumab, and trastuzumab emtansine werediluted with an acetate buffer solution and used at a volume of 10 mL/kgfor intravenous administration to the tail vein of each mouse.

Tumor (ST225; South Texas Accelerated Research Therapeutics (START))excised from a breast cancer patient and maintained at several passagesby transplantation to female nude mice was used in this test. This tumormoderately expressed HER2 (which received a score of 2+ inimmunohistochemical staining)

A tumor section of the solid tumor was subcutaneously transplanted tothe side of the body of each female nude mouse, and the mice wererandomly grouped when the tumor volume reached 100 to 300 mm³. The dateof grouping was defined as Day 0. The antibody-drug conjugate (50),trastuzumab, or trastuzumab emtansine was intravenously administered ata dose of 10 mg/kg to the tail vein of each mouse on Day 0. An acetatebuffer solution administration group was established as a control group.

The results are shown in FIG. 8. The administration of trastuzumab didnot inhibit the growth of the breast cancer ST225 tumor moderatelyexpressing HER2. By contrast, the administration of trastuzumabemtansine or the antibody-drug conjugate (50) remarkably inhibited thegrowth of the tumor.

Evaluation Example 9 Antitumor Test (7)

Tumor (ST910; START) excised from a breast cancer patient and maintainedat several passages by transplantation to female nude mice was used inthis test. This tumor low expressed HER2 (which received a score of 1+in immunohistochemical staining).

A tumor section of the solid tumor was subcutaneously transplanted tothe side of the body of each female nude mouse, and the mice wererandomly grouped when the tumor volume reached 100 to 300 mm³. The dateof grouping was defined as Day 0. The antibody-drug conjugate (50),trastuzumab, or trastuzumab emtansine was intravenously administered ata dose of 10 mg/kg to the tail vein of each mouse on Day 0. An acetatebuffer solution administration group was established as a control group.

The results are shown in FIG. 9. The administration of trastuzumab ortrastuzumab emtansine did not inhibit the growth of the breast cancerST910 tumor low expressing HER2. By contrast, the administration of theantibody-drug conjugate (50) remarkably inhibited the growth of thetumor, demonstrating that the antibody-drug conjugate (50) is effectivefor breast cancer tumor low expressing HER2. This Evaluation Example 9was carried out by the same procedure as Evaluation Example 8.

Evaluation Example 10 Antitumor Test (8)

This test was carried out by the following procedure. EvaluationExamples 11 to 13 were also carried out by this procedure.

Mouse: 5- to 8-week-old female nude mice (Harlan Laboratories Ltd.) weresubjected to the experiment.

Assay and calculation expression: The major axis and minor axis of tumorwere measured twice a week by using an electronic digital caliper, andthe tumor volume (mm³) was calculated. The calculation expression is asshown below.

Tumor volume (mm³)=0.52×Major axis (mm)×[Minor axis (mm)]²

The antibody-drug conjugate, trastuzumab, and trastuzumab emtansine werediluted with an acetate buffer solution and used at a volume of 10 mL/kgfor intravenous administration to the tail vein of each mouse.

Tumor (CTG-0401; Champions Oncology Inc.) excised from a colorectalcancer patient and maintained at several passages by transplantation tofemale nude mice was used in this test. This tumor low or moderatelyexpressed HER2 (which received a score of 1+ or 2+ inimmunohistochemical staining).

A tumor section of the solid tumor was subcutaneously transplanted tothe left side of the body of each female nude mouse, and the mice wererandomly grouped when the tumor volume reached 100 to 300 mm³. The dateof grouping was defined as Day 0. The antibody-drug conjugate (50),trastuzumab, or trastuzumab emtansine was intravenously administered ata dose of 10 mg/kg to the tail vein of each mouse on Day 0. An acetatebuffer solution administration group was established as a control group.

The results are shown in FIG. 10. The administration of trastuzumab ortrastuzumab emtansine did not inhibit the growth of the colorectalcancer CTG-0401 tumor low or moderately expressing HER2. By contrast,the administration of the antibody-drug conjugate (50) remarkablyinhibited the growth of the tumor.

Evaluation Example 11 Antitumor Test (9)

Tumor (CTG-0860; Champions Oncology Inc.) excised from a non-small celllung cancer patient and maintained at several passages bytransplantation to female nude mice was used in this test. This tumormoderately expressed HER2 (which received a score of 2+ inimmunohistochemical staining).

A tumor section of the solid tumor was subcutaneously transplanted tothe left side of the body of each female nude mouse, and the mice wererandomly grouped when the tumor volume reached 100 to 300 mm³. The dateof grouping was defined as Day 0. The antibody-drug conjugate (50),trastuzumab, or trastuzumab emtansine was intravenously administered ata dose of 10 mg/kg to the tail vein of each mouse on Day 0. An acetatebuffer solution administration group was established as a control group.

The results are shown in FIG. 11. The administration of trastuzumab ortrastuzumab emtansine did not inhibit the growth of the non-small celllung cancer CTG-0860 tumor moderately expressing HER2. By contrast, theadministration of the antibody-drug conjugate (50) remarkably inhibitedthe growth of the tumor.

Evaluation Example 12 Antitumor Test (10)

Tumor (CTG-0927; Champions Oncology Inc.) excised from a bile ductcancer patient and maintained at several passages by transplantation tofemale nude mice was used in this test. This tumor highly expressed HER2(which received a score of 3+ in immunohistochemical staining).

A tumor section of the solid tumor was subcutaneously transplanted tothe left side of the body of each female nude mouse, and the mice wererandomly grouped when the tumor volume reached 100 to 300 mm³. The dateof grouping was defined as Day 0. The antibody-drug conjugate (50),trastuzumab, or trastuzumab emtansine was intravenously administered ata dose of 10 mg/kg to the tail vein of each mouse on Day 0. An acetatebuffer solution administration group was established as a control group.

The results are shown in FIG. 12. The administration of trastuzumab didnot inhibit the growth of the bile duct cancer CTG-0927 tumor highlyexpressing HER2. By contrast, the administration of trastuzumabemtansine inhibited the growth of the tumor. Furthermore, theadministration of the antibody-drug conjugate (50) induced theregression of the tumor.

Evaluation Example 13 Antitumor Test (11)

Tumor (CTG-0137; Champions Oncology Inc.) excised from an esophagealcancer patient and maintained at several passages by transplantation tofemale nude mice was used in this test. This tumor highly expressed HER2(which received a score of 3+ in immunohistochemical staining).

A tumor section of the solid tumor was subcutaneously transplanted tothe left side of the body of each female nude mouse, and the mice wererandomly grouped when the tumor volume reached 100 to 300 mm³. The dateof grouping was defined as Day 0. The antibody-drug conjugate (50),trastuzumab, or trastuzumab emtansine was intravenously administered ata dose of 10 mg/kg to the tail vein of each mouse on Day 0. An acetatebuffer solution administration group was established as a control group.

The results are shown in FIG. 13. The administration of trastuzumab didnot inhibit the growth of the esophageal cancer CTG-0137 tumor highlyexpressing HER2. By contrast, the administration of trastuzumabemtansine or the antibody-drug conjugate (50) remarkably inhibited thegrowth of the tumor.

Evaluation Example 14 Antitumor Test (12)

Human ovarian cancer line SK-OV-3 cells highly expressing HER2 purchasedfrom ATCC were suspended in physiological saline, and 4×10⁷ cells weresubcutaneously transplanted to the right side of the body of each femalenude mouse to prepare SK-OV-3 solid tumor. Thereafter, this solid tumorwas maintained at several passages by transplantation to female nudemice and used in this test.

A tumor section of the solid tumor was subcutaneously transplanted tothe right side of the body of each female nude mouse, and the mice wererandomly grouped when the tumor volume reached 100 to 300 mm³. The dateof grouping was defined as Day 0. The antibody-drug conjugate (50),trastuzumab, or trastuzumab emtansine was intravenously administered ata dose of 10 mg/kg to the tail vein of each mouse on Day 0. Aphysiological saline administration group was established as a controlgroup.

The results are shown in FIG. 14. The administration of trastuzumab didnot inhibit the growth of the SK-OV-3 tumor. By contrast, theadministration of trastuzumab emtansine or the antibody-drug conjugate(50) remarkably inhibited the growth of the tumor.

Free Text of Sequence Listing

SEQ ID NO: 1—Amino acid sequence of a heavy chain of the humanizedanti-HER2 monoclonal antibodySEQ ID NO: 2—Amino acid sequence of a light chain of the humanizedanti-HER2 monoclonal antibody

1. An antibody-drug conjugate, wherein a linker and an antitumorcompound represented by the following formula and anti-HER2 antibody areconnected:-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—(NH-DX)wherein -(Succinimid-3-yl-N)— has a structure represented by thefollowing formula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1, and (NH-DX) represents agroup represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is theconnecting position.
 2. The antibody-drug conjugate according to claim1, wherein an average number of units of the selected one drug-linkerstructure conjugated per antibody is in a range of from 2 to
 8. 3. Theantibody-drug conjugate according to claim 1, wherein an average numberof units of the selected one drug-linker structure conjugated perantibody is in a range of from 3 to
 8. 4. A drug containing theantibody-drug conjugate according to claim 1 or a salt thereof.
 5. Anantitumor drug and/or anticancer drug containing the antibody-drugconjugate according to claim 1 or a salt thereof.
 6. A method oftreating cancer in an individual comprising administering to anindividual with cancer the drug according to claim 5, wherein the canceris lung cancer, urothelial cancer, colorectal cancer, prostate cancer,ovarian cancer, pancreatic cancer, breast cancer, bladder cancer,gastric cancer, gastrointestinal stromal tumor, uterine cervix cancer,esophageal cancer, squamous cell carcinoma, peritoneal cancer, livercancer, hepatocellular cancer, colon cancer, rectal cancer, colorectalcancer, endometrial cancer, uterine cancer, salivary gland cancer,kidney cancer, vulval cancer, thyroid cancer, penis cancer, leukemia,malignant lymphoma, plasmacytoma, myeloma, or sarcoma.
 7. Apharmaceutical composition containing the antibody-drug conjugateaccording to claim 1 or a salt thereof as an active component, and apharmaceutically acceptable formulation component.
 8. A method oftreating cancer in an individual comprising administering to anindividual with cancer the pharmaceutical composition according to claim7, wherein the cancer is lung cancer, urothelial cancer, colorectalcancer, prostate cancer, ovarian cancer, pancreatic cancer, breastcancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor,uterine cervix cancer, esophageal cancer, squamous cell carcinoma,peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer,rectal cancer, colorectal cancer, endometrial cancer, uterine cancer,salivary gland cancer, kidney cancer, vulval cancer, thyroid cancer,penis cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, orsarcoma.